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Ischemia/reperfusion (IR) injury results from a prolonged ischemic insult followed by restoration of blood perfusion. It affects all oxygen dependent cells that rely on an uninterrupted blood supply. These aerobic cells require mitochondrial oxidative phosphorylation for their energy supply. Consequently all aerobically metabolizing tissues and organs are potential targets of IR injury.1
Significant liver IR injury during surgical resection is commonly a consequence of prolonged portal triad clamping followed by reperfusion, performed as an elective preplanned procedure or as an emergent maneuver, to control excessive bleeding from the cut hepatic surface.2 Transplantation of the liver can also lead to IR injury. Here, the damage is sustained during cold preservation of the liver following explantation from the donor, and during subsequent warm reperfusion at implantation into the recipient.3 In non–heart beating transplantation, there is additional warm ischemic damage until hepatic cold perfusion is commenced. Systemic low flow states and hypoxia such as is encountered in hemorrhage,4 sepsis, congestive cardiac failure, trauma, and respiratory failure may also lead to liver IR injury.5
The degree of IR injury sustained is dependent on the length and method of ischemia applied to the liver, as well as the background liver condition. Patients who undergo short intermittent periods of ischemia sustain less liver dysfunction compared to those receiving a continuous period of ischemia.6 This is especially the case in patients with chronic liver disease.6 Animal studies also support the notion that the background liver parenchymal condition significantly determines the degree of tolerance to IR. Animal models of chronic liver disease exhibit worse liver damage when subjected to IR compared to those with normal livers.7, 8 Other determinants of liver susceptibility to IR injury include age (older livers exhibit more damage compared to young ones)9 and sex (in animal models, males are less tolerant of ischemic insults than are females; however there is insufficient evidence for a firm clinical recommendation on the role of sex in liver IR injury).10 Systemic and localized neoadjuvant chemotherapy is increasingly being used to down-stage liver tumors prior to surgical resection. There is increasing evidence that, contrary to instinctive beliefs, neoadjuvant chemotherapy does not increase liver susceptibility to IR injury.11, 12 In fact, through suppression of inflammatory pathways, chemotherapy may attenuate the IR injury.11, 12
In the mildest form of IR injury sustained during liver resections, liver aminotransferase enzymes are elevated in the circulation. More severe insults lead to clinical liver dysfunction and progressive failure. Direct IR to the liver can also result in distant organ dysfunction due to remote IR injury. Lungs,13 heart,14 kidneys,15 and blood vessels16 have all been shown to sustain remote dysfunction secondary to direct liver IR. Substantial IR injury sustained during liver transplantation increase the incidence of primary graft nonfunction, primary graft dysfunction, and biliary strictures.17 Hepatic nonfunction and intrahepatic biliary strictures in transplantation, and failure of the remnant liver in resectional surgery usually necessitate transplantation, placing an even greater pressure on the limited number of donor livers.
Liver IR injury is a complex process involving numerous cell types and molecular mediators in various pathways. The cellular damage occurs during both the ischemic and reperfusion phases. The end result is cellular death via a combination of apoptosis and necrosis. Warm ischemic damage arises when the blood supply to the liver is interrupted at normal body temperature, as is the case during liver resections. Cold ischemic damage arises during cold perfusion and storage of livers in transplantation. This review is an up-to-date summary of recent discoveries on the cellular and molecular mechanisms that mediate the inflammatory response to liver IR injury. The discussion begins by reviewing the function of each of the main cellular types within the liver that participate in IR injury. This is followed by a description of the role of humoral factors, endogenous danger signals, and mitochondria in liver IR injury.
During the ischemic phase as a result of glycogen consumption and lack of oxygen supply, Kupffer cells, sinusoidal endothelial cells (SECs), and hepatocytes suffer with lack of adenosine triphosphate (ATP) production.18 The lack of ATP leads to failure of the sodium/potassium ATP-dependent plasma membrane pump (Na+/K+ adenosine triphosphatase [ATPase]) and subsequent intracellular Na+ accumulation, edema, and swelling. Kupffer cell and SEC swelling combined with an increase in the vasoconstrictors endothelin and thromboxane A2 and a decrease in the vasodilator nitric oxide19 lead to sinusoidal narrowing. In addition, on reperfusion there is increased adhesion and aggregation of neutrophils and platelets in the sinusoids. The end result is significant reduction of microcirculatory blood flow on reperfusion, including some areas with complete absence of blood flow, which is known as “no-reflow”20 (Fig. 1).
Kupffer cells are the liver-resident macrophages. They play a key role in initiating and propagating cellular damage and death in IR injury.21 Kupffer cells are activated during the ischemic phase and even more so on reperfusion. On activation, they produce reactive free radicals, specifically, reactive oxygen species (ROS) and proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β).22 These ROS and cytokines have widespread effects in the initiation and propagation of IR injury. Their main role is to recruit and activate circulating neutrophils on reperfusion. In addition they recruit and activate CD4+ T lymphocytes,23 activate SECs leading to expression of cell-surface adhesion molecules and contribute to their damage,23 stimulate hepatocytes to produce ROS and contribute to their damage,24 and stimulate platelet adhesion to SECs.25 Kupffer cells are activated by the complement system during direct or remote IR injury of the liver.26, 27 Additional Kupffer cell activation is achieved by interferon-γ (IFN-γ) produced by CD4+ T cells and natural killer T cells28, 29 (Fig. 2).
Activated SECs, hepatocytes, and neutrophils express cell-surface adhesion molecules. On neutrophils, L-selectin and β2-integrins (CD11b/CD18) adhere to intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) on the surface of SECs and hepatocytes30 (Fig. 2). In liver IR injury however, early neutrophil accumulation in the sinusoids mainly results from mechanical trapping by the swollen, constricted, platelet-adhering sinusoids. In response to chemokines from activated and injured hepatocytes, sinusoidally accumulated neutrophils use the surface-expressed adhesion molecules to bind their SEC counterpart and migrate across the endothelium into the parenchyma. The binding of CD11b/CD18 on the neutrophils to ICAM-1 and VCAM-1 on the hepatocytes triggers an enhancement of ROS production through the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system as well as degranulation of cytoplasmic vesicles containing various enzymes that assist in the degradation of the extracellular matrix and dead hepatocytes, and damage viable hepatocytes.30
The liver contains all the subtypes of lymphocytes as resident cells. These comprise B cells, CD8+ and CD4+ T cells (conventional T lymphocytes), natural killer T (NKT) cells, and γδ T cells (unconventional T lymphocytes).31 CD4+ and to a lesser extent NKT lymphocytes play a key role in liver IR injury. CD4+ cells accumulate in the liver within 1 hour of reperfusion following IR, preceding any neutrophil accumulation.32 They are recruited to the liver and activated by various Kupffer cell-derived products (Fig. 2). These include chemokines, ROS, cytokines, and matrix metalloproteinase 9.23, 33, 34 CD4+ cells are important contributors in neutrophil recruitment into the postischemic liver.32 CD4+ lymphocytes stimulate neutrophil accumulation through production of the chemotactic agent IL-17.32 In addition, CD4+ cells produce IFN-γ, which activates Kupffer cells to produce TNF-α and IL-1, and hepatocytes to produce chemokines.28 Thus CD4+ T lymphocytes and Kupffer cells reciprocally activate one another.23 The end result of hepatic CD4+ cell recruitment and activation postischemia is hepatocyte and SEC damage with microvascular perfusion defects.35 NKT cells are recruited and activated into the postischemic liver on a time scale similar to CD4+ cells.32 NKT cell activation is dependent on interaction with CD1d antigen-presenting molecules expressed on hepatocytes and antigen presenting cells within the liver.29, 36 CD1d presents either self or foreign glycolipid antigens to NKT cells. Activated NKT cells are capable of damaging the liver directly and of producing IFN-γ with subsequent Kupffer cell and hepatocyte activation.29, 36
Two important groups of humoral factors participate in liver IR injury: the complement system and cytokines. The complement system comprises approximately 30 soluble and membrane-bound proteins. Three distinct pathways are known to activate the complement cascade; the classical, alternative, and mannose-binding lectin pathways. All 3 pathways contribute to complement activation in liver IR injury.37 Once activated in IR injury, complement leads to liver damage either directly by lysing liver cells through the formation of a membrane attack complex in the plasma membrane,38 or by recruiting neutrophils and activating both neutrophils and Kupffer cells20 (Fig. 2). Complement activation has also been implicated in the pathogenesis of remote IR injury. For example, in rodents, bilateral hindlimb IR injury leads to remote liver IR injury by activation of Kupffer cells via complement factor C5a.27, 39 Direct liver IR injury on the other hand, results in remote endothelial damage in the lungs and small bowel in the mouse, which is significantly attenuated in mice transgenically overexpressing the physiologically occurring complement inhibitor called C1 inhibitor.40 C1 inhibitor inhibits complement factor C1 and, therefore, the classical pathway of complement activation. These observations implicate C1 (classical pathway) as a mediator of liver IR injury–induced remote organ injury.40
Cytokines may play a proinflammatory or an anti-inflammatory role in liver IR injury. An extensive volume of literature is available on the function of cytokines in liver IR injury, but only a summary of the actions of the main cytokines involved in liver IR injury is provided in this review. TNF-α is the central component of the proinflammatory cytokine cascade in liver IR injury. It is produced by Kupffer cells and acts both locally in a paracrine fashion, and remotely as an endocrine mediator. It is a crucial effecter of remote organ damage following hepatic IR.41 A number of endogenous proinflammatory and anti-inflammatory molecules may stimulate or inhibit, respectively, the expression of TNF-α in liver IR injury. Table 1 lists those molecules that are well-established in fulfilling these functions. The up-regulation of TNF-α in liver IR injury leads to liver damage. TNF-α binds to specific TNF-receptors (TNF-R) on the surface of hepatocytes. This results in increased production of the chemokine epithelial neutrophil activating protein-78 (ENA-78) and ROS, as well as activation of nuclear factor kappa B (NF-κB) and the mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK).42 In addition, TNF-α up-regulates expression of the adhesion molecules ICAM-1, VCAM-1, and P-selectin.41, 43 Through various mechanisms, all these molecules are able to recruit and activate neutrophils into the postischemic liver. Furthermore, JNK and ROS are capable of direct cellular damage within the liver.42
Table 1. Stimulators and Inhibitors of TNF-α Expression in Hepatic IR Injury
Effect on TNF-α Expression
Transcription Factor Intermediate
IL-6 has been shown to inhibit TNF-α expression and activate STAT-3 in separate experiments.
JNK is not a transcription factor but an intracellular transduction pathway that phosphorylates transcription factors, among other functions.
The role of other important cytokines in liver IR injury is summarized in Table 2. The expression of these cytokines is under the control of a variable group of upstream regulators. Downstream, the cytokines influence the expression of various molecules that ultimately lead to either protection from, or exacerbation of liver IR injury. Each of these molecules belongs to a group that shares similar functions. There are transcription factors (activator protein-1 [AP-1], heat shock factor, NF-κB, signal transducer and activator of transcription-3 [STAT-3], and STAT-6), antioxidants (superoxide dismutase [SOD], glutathione [GSH]), adhesion molecules (ICAM-1, E-selectin), inflammation-stimulated inducible enzymes (COX-2, inducible NO synthase [iNOS], and HO-1). Intracellular signaling molecules (Akt), antiapoptotic proteins (Bcl-2/Bcl-x exert their effect by altering the mitochondrial membrane permeability), and chemokines (CINC, ENA-78, IP-10, MCP-1, MIG, MIP-2). The latter are chemotactic cytokines that direct and regulate the accumulation of neutrophils, monocytes/macrophages, and T lymphocytes into the postischemic liver.
Table 2. The Role of Important Cytokines in Liver IR Injury
Downstream Mediator(s) of Effect
Summary Effect on Liver
IL-12 and IL-23 are grouped together because they share structural homology and exhibit similar actions in liver IR injury.
1. Endogenous expression with hepatic IR injury: activation of VEGF receptor and cytosolic SRC tyrosine kinases, ↑ expression of TNF-α, IFN-γ, MCP-1, and E-selectin leading to hepatic accumulation of T cells, macrophages, and neutrophils.
1. Endogenous expression increases hepatic IR injury.
2. Exogenous administration: iNOS up-regulation.
2. Exogenous administration reduces hepatic IR injury.
The foregoing discussion on the role of the various cellular and humoral players (summarized in Fig. 2 and Tables 1 and 2) that contribute to liver IR injury illustrates the complex communication networks used by the immune and nonimmune systems to initiate and propagate IR injury. However, we are only just beginning to understand these interactions and further research is warranted to further unravel how immune activation leads to metabolic disturbances.
ENDOGENOUS DANGER SIGNALS
These comprise the danger-associated molecular patterns (DAMPs) which act as normal physiological constituents of cells or the extracellular environment. DAMPs are either released passively by necrotic cells or the damaged extracellular matrix, or are actively secreted by stressed and injured cells. They have the ability to initiate and propagate the inflammatory response in an analogous manner to the pathogen-associated molecular patterns (PAMPs). PAMPs are conserved molecular motifs found in various prokaryotic pathogens but not in mammals, and are therefore recognized as foreign by the cells of the innate immune system in the host, leading to the initiation of an immune response.44 Examples of PAMPs include lipopolysaccharide from gram-negative bacteria, lipoteichoic acid from gram-positive bacteria, and double-stranded RNA from viruses.45 Examples of DAMPs released during liver IR injury include the nuclear transcription factor high mobility group box-1 (HMGB-1),46, 47 the cytoplasmic Ca2+ regulator S100,48 the cell matrix component hyaluronic acid,49 urate,50 ATP,50 and DNA.51 Other clinical conditions that may result in increased expression and release of DAMP molecules include sterile inflammatory conditions such as burns and cancer, and infectious states such as severe sepsis and localized purulent infections.44 Both DAMPs and PAMPs exert their effects by binding to a group of receptors termed pattern recognition receptors (PRRs), either on the surface of or in the cytoplasm of mammalian cells. PRRs are constitutively expressed on cells of the innate immune system and initiate an inflammatory response upon binding DAMP or PAMP molecules. Two classes of PRRs have been shown to initiate and propagate the inflammatory responses in liver IR injury: the toll-like receptors (TLRs), specifically TLR-4, and the receptor for advanced glycation end products (RAGE).
The TLRs are type 1 transmembrane proteins containing an extracellular amino-terminus and an intracellular carboxy-terminal domain. The latter includes an IL-1 and an IL-18 receptor–like signaling motif called Toll–IL-1 receptor (TIR) homology domain.52 TLRs that detect DAMP released during sterile inflammatory conditions of the liver provide an important link between liver parenchymal damage and the activation of the immune system. Eleven human TLRs (TLR-1 to TLR-11) have been identified. However, in liver IR injury, TLR-4 has been by far the most extensively investigated. The general and liver IR injury–specific TLR-4 signaling pathways are summarized in Fig. 3. Other TLRs use similar pathways of signal transduction. The general model of TLR-4 mechanism of action involves engagement of the receptor by its ligand(s) which triggers an intracellular signaling cascade that culminates in the up-regulation of proinflammatory cytokines, chemokines, and other mediators of inflammation.52 The cascade begins with the TIR domain on the cytoplasmic portion of the receptor recruiting and activating 1 or more TIR-containing intracellular adaptors. The type of adaptor recruited broadly defines the subsequent intracellular signaling pathway involved. The adaptor recruited may be myeloid differentiation factor 88 (MyD88)-dependent, which is also common to all other human TLR except TLR-3, or MyD88-independent adaptors. The latter includes TIR domain–containing adaptor protein (TIRAP), TIR domain–containing adaptor inducing interferon β (TRIF), and TRIF-related adaptor molecule (TRAM).53 The end result of MyD88-dependent signaling is proinflammatory cytokine gene transcription; the end result of the MyD88-independent pathway is induction of type 1 IFN and subsequent up-regulation of the leukocyte chemoattractant IP-10.53, 54 Both signaling pathways act to propagate the inflammatory response. For TLRs that are capable of transducing their signal through more than 1 pathway such as TLR-4, the factor that determines which pathway is recruited every time the receptor is activated is the cell type. For example, MyD88 signaling exclusively transduces TLR-4 activation in the endothelium because these cells lack TRAM expression.52 However, within a defined cell type, the specificity of TLR-mediated signaling is determined by a combination of the TLR type, the intracellular adaptor, and the intracellular signaling pathway.54 In addition to the well-described role of TLR-4 activation in liver IR injury, recent published work shows TLR-9 receptor activation by HMGB-1 and DNA, both released by necrotic hepatocytes, play an important role in mediating liver IR injury.51
Among the DAMPs released during liver IR injury, HMGB-1 is the best characterized. HMGB-1 levels are significantly elevated in the serum and liver tissue after IR and lead to liver injury.47, 55 All nucleated cells within the liver possess HMGB-1 and will release it upon necrosis, thereby permitting it to interact with and activate TLRs. Signaling molecules in the MyD88-dependent and MyD88-independent pathways have been shown to mediate TLR-4 activation in liver IR injury (Fig. 3). These include the transcription factors NF-κB, AP-1, IRF-3, and STAT-1; the MAP kinases P38, JNK, and ERK; Akt; ROS; and cytokines and chemokines.47, 56-61 IP-10, a leukocyte chemoattractant molecule that is the final consequence of the MyD88-independent pathway, has been shown to activate ERK in liver IR injury, which is associated with significant hepatocellular damage and proinflammatory cytokine release.61 Furthermore, IP-10 knockout mice subjected to liver IR show reductions in ERK activation compared to wild-type animals, associated with significant attenuation in liver IR injury and reduction in proinflammatory cytokine release.61 Therefore, IP-10 forms a positive feedback loop that activates ERK and thereby provides a link between the MyD88-dependent and MyD88-independent pathways (Fig. 3). TLR receptors on nonparenchymal liver cells derived from the bone marrow are preferentially activated in liver IR injury and result in hepatocellular damage. The hepatocytes do not undergo TLR activation directly by DAMP molecules but respond to inflammatory mediators produced from the nonparenchymal cells that are directly stimulated by DAMPs.62 For example, it has been shown that TLR-4 receptors on Kupffer and dendritic cells58, 63 and TLR-9 receptors on neutrophils51 are activated during liver IR and result in hepatocellular damage. The MyD88-independent pathway using IRF3–type 1 IFN in hepatic IR is down-regulated by HO-1. HO-1 induction blocks the expression of type 1 IFN, the subsequent activation of STAT-1, and the expression of downstream IP-10.56, 57 Furthermore, HO-1 even leads to a reduction in TLR-4 protein levels.64 The mechanisms described hitherto have all been elucidated in direct liver IR models. However, in an animal model of hemorrhagic shock followed by resuscitation, which is synonymous with systemic IR injury, serum and liver HMGB-1 levels are significantly elevated, causing activation of TLR-4s on circulating polymorphonucleocytes. Activated TLR-4s increase ROS production through the NADPH oxidase system (Fig. 3). This is achieved through the sequential activation of MyD88-IRAK4-p38 MAPK or MyD88-IRAK4-Akt downstream of TLR-4.65 In addition, trauma hemorrhage followed by resuscitation results in engagement of TLR-2, TLR-4, and TLR-9 in Kupffer cells with subsequent p38 and JNK activation.66 Thus, in hepatic IR injury, the signaling pathway(s) mediating the activation of a PRR will at least be partly dependent on the mechanism of the IR.
The receptor for advanced glycation end products (RAGE) is the second family of PRRs that is involved in liver IR injury (Fig. 4). Within the liver, RAGE is mainly expressed on dendritic cells and to a lesser extent on Kupffer cells.67, 68 Hepatic IR leads to increased expression of RAGE and engagement of these receptors by endogenous DAMP such as HMGB-1 with consequent hepatocellular injury.67, 68 HMGB-1 binding to RAGE in the setting of liver IR leads to a signaling cascade involving the activation of JNK/ERK, which increases the expression and activation of the inducible transcription factor early growth response-1 (Egr-1). Egr-1 acts as a coordinating up-regulator of divergent gene families affected by stress that in the setting of hepatic IR includes MIP-2, which functions to recruit immune cells into the postischemic liver.68 Engagement of RAGE by other DAMP results in the activation of other intracellular mediators of inflammation such as p38 MAPK, and the transcription factors STAT-3 and AP-1, with a consequent increase in proinflammatory cytokines such as TNF-α67 (Fig. 4).
In IR injury, the mitochondrion participates in various pathophysiological processes. There is failure of ATP production as a consequence of disruption of oxidative phosphorylation caused by the generation of and damage by reactive oxygen and nitrogen species (ROS and RNS, respectively). Cytosolic ionic ([Ca2+] [Na+] [H+]) disturbances lead to mitochondrial ionic disturbances, and vice versa, with consequent plasma and mitochondrial membrane damage, including the formation of mitochondrial permeability transition (MPT) pores.
ROS AND RNS
The main intracellular mechanisms for producing ROS during IR are the xanthine oxidase, mitochondrial respiratory chain, and NADPH oxidase systems.69-71 The main ROS generated are superoxide radical (O), hydrogen peroxide (H2O2), hypochlorous acid (HCLO), and hydroxyl radical (·OH). The latter is the product of O or H2O2 interaction with certain transition metals, for example iron or copper.71 The biologically important RNS include nitric oxide (NO), nitrogen dioxide (NO2), dinitrogen trioxide (N2O3), and peroxynitrite (ONOO−). These are produced when NO reacts with O and molecular oxygen.72 For a detailed description of the pathways involved in ROS and RNS generation in IR injury, the reader is referred to recent published reviews.71, 73
ROS and RNS contribute to injury of the various cell types found in the liver, leading to both apoptotic and necrotic cell death. ROS cause oxidative damage to membrane lipids (lipid peroxidation), especially polyunsaturated fatty acids, which result in disturbances in ion homeostasis, cell swelling, and death.70 The damage sustained is not limited to the plasma membrane but also extends into the intracellular compartment to encompass all membrane-bound organelles, including the mitochondrion and the nucleus. In addition, within the mitochondria, ROS can cause oxidative damage to the enzyme complexes of the respiratory chain, leading to failure of ATP production and release of cytochrome C into the cytosol, triggering apoptosis.74, 75 Within the nucleus the DNA can be oxidatively damaged resulting in failure of protein transcription and translation. Antiproteases can also sustain ROS damage, leading to their inactivation, which allows inappropriate activation of proteases to inflict cellular damage. ROS can also activate the redox-sensitive transcription factors, namely NF-κB and AP-1.42, 76 Among their functions, NF-κB up-regulates proinflammatory cytokines like TNF-α,77 and AP-1 promotes hepatocyte apoptosis through cytochrome C release and caspase-3 activation.42 ROS generation during IR may also stimulate further ROS generation from neighboring mitochondria. This recently described phenomenon is termed “ROS-induced ROS release” (RIRR), and occurs when ROS production reaches a threshold level that causes the MPT pore or the inner membrane anion channels, or both, to open resulting in loss of the mitochondrial membrane potential with subsequent cessation of electron flow in the respiratory chain and increased ROS production by complexes I through III. With a disrupted mitochondrial membrane, a large amount of ROS is released into the cytosol that may induce RIRR in adjacent mitochondria. Thus, this mechanism of ROS production acts as a self-perpetuating cycle leading to mitochondrial and cellular destruction. The exact contribution of RIRR to ROS generation in liver IR injury is unknown and requires further research.
RNS inflict their damage by similar mechanisms described for ROS. In addition, they cause posttranslational protein modifications that may result in the inactivation or introduction of a new function to a protein.78 A detailed discussion of the molecular biology of RNS and their redox reactions is beyond the scope of this review but can be found elsewhere.79
Perturbations in the homeostatic concentrations of Ca2+, Na+, and H+ occur in liver IR and may result in cellular death if excessive.
Ca2+ is mainly found in 3 cellular compartments, the cytosol, the mitochondria, and the endoplasmic reticulum (ER). The interplay of Ca2+ movement across the membranes of these structures determines the temporal and spatial distribution of [Ca2+] in the 3 compartments. In order to understand the pathological disturbances of Ca2+ homeostasis in liver IR injury, it is important to have a good comprehension of the various mechanisms that control Ca2+ movement across the membranes of the 3 Ca2+-regulating cellular compartments (Table 3).
Table 3. Physiological Ca2+ Pathways in the Plasma, Mitochondrial, and ER Membranes of Hepatocytes
Main Regulating Signal(s) of Ca2+ Movement
Direction of Ca2+ Movement
Role in IR
Ligand-gated Ca2+ channels
Extracellular signaling molecule (eg, hormone, growth factor) binds to receptor on extracellular domain of Ca2+ channel
Extracellular signaling molecule (eg, hormone, growth factor) binds to G protein–coupled receptor or tyrosine kinase–coupled receptor, leading to generation of second messengers that bind to the cytoplasmic domain of the Ca2+ channel that is separate from the receptor
The Na+ and Ca+ gradients across the plasma membrane. In the heart, the Na+/Ca+ exchanger is regulated by phosphorylation by protein kinase A (PKA) and C (PKC), which in turn are activated by numerous extracellular signals (eg, hormones)
Normally extrudes Ca+ into extracellular space but may act in reverse if Na+/Ca+ gradient is altered, eg, if [Na+]extracellular is decreased massively
Extracellular signaling molecule (eg, vasopression, ADP, angiotensin II, noradrenaline) binds to plasma membrane G protein–coupled receptor leading to production of PLC. PLC produces IP3 and diacylglycerol (DAG) which stimulate IP3 and ryanodine receptors, resulting in Ca2+ release into the cytosol
Cytosolic [Ca2+] and mΔΨ. The mP2Y receptors regulate Ca2+ flow through the uniporter. Specifically, mP2Y1 are activated by ADP and AMP and lead to increased mCa2+ uptake; mP2Y2 are activated by ATP and leads to inhibition of mCa2+ uptake. Both P2Y receptors use PLC to regulate mCa2+ uptake through the uniporter
Liver IR injury leads to disturbances in the Ca2+ regulating mechanisms in the plasma, ER, and inner mitochondrial membranes (Fig. 5). The consequence is Ca2+ overload in the cytosol and mitochondrial matrix.80 The rise in cytosolic [Ca2+] results from a combination of increased Ca2+ entry across the plasma membrane as well as Ca2+ release from ER stores. This is brought about by the activation of SOC channels in the plasma membrane and ryanodine receptors in the ER membrane.81, 82 How these channels and receptors are activated is not clear. However, there is evidence that ROS and RNS can activate some Ca2+-permeable nonselective cation channels, the so-called transient receptor potential (TRP) channels.83 TRP channels include some ligand gated, receptor activated, and stretch activated Ca2+ channels (Table 3).84 Specifically, the TRPM7 channel subtype is found in liver cells, and ROS and RNS can activate these channels, leading to Ca2+ influx from the extracellular space into the cytosol.83, 85 In addition, cold ischemic injury inhibits both the plasma membrane and ER Ca2+ ATPases, which normally function to extrude cytosolic Ca2+ into the extracellular space and ER lumen respectively, thereby compounding the rise in [Ca2+]cytosol.86
The rise in cytosolic Ca2+ during liver IR injury leads to a secondary rise in mitochondrial Ca2+ (mCa2+).80 This is a consequence of stimulation of the mitochondrial Ca2+ uniporter.87 How these Ca2+-selective channels are activated in IR injury has not been fully elucidated but probably involves the PLC-dependent mP2Y-like receptors that regulate Ca2+ flow through the mCa2+ uniporter.88, 89 mP2Y1 are activated by ADP and AMP and lead to stimulation of Ca2+ uptake by the uniporter. The mP2Y2 are activated by ATP, ADP, and AMP and lead to the inhibition of the uniporter. During the ischemic phase of IR injury, there is a relative decrease in [ATP]cytosol and an increase in [AMP and ADP]cytosol resulting in an overall increase in the activity of the Ca2+ uniporter and therefore in mitochondrial [Ca2+].90 This increase in mitochondrial [Ca2+] results in a reduction of the mitochondrial transmembrane potential (mΔΨ). In response, and in order to maintain mΔΨ, ATP-synthase reverses its activity and hydrolyzes ATP to provide energy for the various ionic pumps in the mitochondrial membrane that maintain the mitochondrial potential. This results in partial restoration of mΔΨ, which in turn leads to a further increase of Ca2+ inflow through the mCa2+ uniporter. Therefore, a cycle of increasing mCa2+ overload and ATP depletion (as the mitochondria become a net consumer of ATP during ischemia as a consequence of the reversed activity of ATP synthase) is established.80 The mCa2+ overload leads to Bax translocation from the cytosol to the mitochondria and subsequent MPT pore formation, cytochrome C release, and cellular death.87, 91
Sodium (Na+) and hydrogen (H+) ions also have important functions in determining the liver's response to IR insults. Hepatic ischemia results in intracellular acidosis through anaerobic respiration.92, 93 To restore intracellular pH toward normal, the liver cells activate the Na+/H+ exchanger (NHE) and the Na+/HCO cotransporter (NHCT) (Fig. 5). The NHE extrudes H+ into the extracellular space in exchange for Na+ inflow into the cell. The NHCT transports Na+ and HCO intracellularly. Both these mechanisms of correcting the ischemically induced intracellular acidic pH result in intracellular Na+ accumulation. It is the increase in intracellular [Na+] ([Na]i) secondary to intracellular acidosis that contributes to cellular death. Intracellular acidosis per se is actually cytoprotective.94 Support for this finding comes from 5 experimental observations. First, blocking the function of the NHE and the NHCT protects against hepatic IR injury.93, 95 Second, in vitro experiments using Na+-free incubation buffers results in hepatocyte and SEC protection by inhibiting the rise in [Na+]i.94 Third, IPC protects hepatocytes by the activation of vacuolar (H+)-ATPase (V-ATPase) which lead to H+ extrusion from the cells, with a subsequent decrease in intracellular [Na+].96 Fourth, maintenance of an acidic pH at reperfusion results in a decrease in [Na+]i and cellular protection, a phenomenon known as the “pH paradox”.95 Fifth, ischemia-induced hepatocyte death is a result of ATP depletion with subsequent inhibition of the Na+/K+ ATPase and an increase in [Na+]i.95 The question as to how increased [Na+]i promotes, and decreased [H+]i inhibits, cellular death in liver IR injury is not fully resolved. Two experimental observations have been reported that shed light on this. The first is that [Na+]i accumulation does not lead to hepatocyte death by promoting cellular swelling94; the second is that maintenance of an acidic pH at reperfusion prevents the formation of MPT pores.92 Further research is needed to fully explain how [Na+]i and [H+]i mediate their respective action in hepatic IR injury.
MITOCHONDRIAL PERMEABILITY TRANSITION
The mitochondria is surrounded by a double membrane. The outer mitochondrial membrane (OMM) is significantly more permeable than the inner mitochondrial membrane (IMM). This is due to the presence of voltage-dependent anion channels (VDAC) in the OMM that nonselectively permit the passage of solutes up to 5 kDa size. Under normal physiological conditions, the IMM is permeable to solutes only through specific exchangers, channels, and transporters. However, under certain circumstances such as IR injury, the IMM becomes nonselectively permeable to molecules up to 1500 Da, resulting in mitochondrial depolarization and collapse of the mΔΨ, uncoupling of oxidative phosphorylation, and mitochondrial swelling due to colloid osmotic forces that lead to the release of proapoptotic factors such as cytochrome C. These changes are known as the MPT.92 The MPT is due to the formation and opening of pores, with an average diameter of 3 nm when fully opened, through the IMM. It used to be thought that these pores are formed from VDAC in the OMM, adenine nucleotide transporter (ANT, which is responsible for exchanging ADP/ATP across the IMM) in the IMM, and cyclophilin D (CycD, a mitochondrial matrix protein) in the matrix. However, this model did not explain experimental observations such as why ANT knockout mice still undergo MPT.97 The current prevailing hypothesis is that the MPT is formed from integral mitochondrial membrane proteins, that as a consequence of modifications by ROS and by reactive chemicals, lead to exposure of hydrophilic residues in the protein to the hydrophobic membrane environment, and as a result the proteins aggregate at these hydrophilic residues to form channels that conduct aqueous solutes98 (Fig. 6). The identity of proteins in the MPT pores is not known for certain but is thought to include misfolded ANT and anion transporters such as aspartate-glutamate and phosphate carriers.99 The MPT pore protein composition is not consistent but depends on the relative abundance of the misfolded proteins. Therefore, even in ANT-deficient mitochondria, MPT pore formation can still occur.99 Moreover, 2 conductance modes of MPT pores are thought to be operational depending on the number of misfolded proteins and therefore on the number of pores formed. In the regulated conductance mode, the MPT pores associate with chaperone-like proteins, including CycD on the inner surface of the IMM (Fig. 6). These proteins serve to inhibit conductance through the MPT pores. When the number of newly formed MPT pores exceeds the number of chaperones available, then unregulated pore opening occurs.100 CycD confers Ca2+, ROS, and cyclosporine A sensitivity on pore conductance.101 A rise in matrix [Ca2+] increases, and cyclosporine A inhibits pore opening.
Many other factors influence opening of and therefore conductance through the pores.102 Aside from Ca2+, other divalent cations such as Mg2+, Sr2+, and Mn2+ inhibit pore opening. H+ (and therefore acidic pH) inhibit pore opening by the reversible protonation of histidyl residues, thereby explaining why maintenance of acidosis at reperfusion of ischemic hepatocytes significantly decreases cellular death. ROS induce pore opening. Inorganic phosphate (Pi) plays a dual role in regulating MPT pore opening. In the mitochondrial matrix, Pi binds H+ and Mg2+ blocking their pore-inhibitory effect, resulting in pore opening. On the other hand Pi can bind to MPT pores directly leading to inhibition of pore opening only when CycD has been ablated or in the presence of cyclosporine A which inhibits CycD activity. This is because it is thought that active CycD normally binds to the Pi-binding sites on the MPT pores, thereby preventing Pi binding. In the absence or inactivation of CycD, Pi binds to and inhibits the opening of MPT pores.102
The end result of MPT depends on the number of mitochondria affected and therefore cellular ATP levels. When mitochondria within a hepatocyte are afflicted by MPT, these mitochondria are depolarized and are permanently damaged. When the number of these dysfunctional mitochondria is small, the hepatocyte removes these mitochondria by the process of mitophagy.103 Mitophagy is the process of sequestration of mitochondria into autophagosomes and their delivery to lysosomes for degradation. This is an important process for cells, as damaged mitochondria are a source of accelerated ROS production and ATP consumption.104 As the number of mitochondria undergoing MPT rises, with relative preservation of cellular ATP levels, apoptotic cell death occurs due to cytochrome C release and subsequent formation of the ATP-dependent apoptosome. When the majority of the hepatocyte's mitochondria undergo MPT, the ATP levels plummet, causing cell death by necrosis.92, 105 In summary, with an increasing number of mitochondria undergoing MPT, the hepatocyte's graded response is: mitophagy → apoptosis → necrosis. Whereas in the preceding discussion, the majority of the experimental data support the observation that IR resulting in MPT leads to mitophagy, there has also been recent work showing that, on the contrary, anoxia/reoxygenation of cultured hepatocytes that results in MPT does not cause mitophagy but leads to accumulation of dysfunctional mitochondria,106 which are the source of increased ROS production and subsequent mitochondrial damage, including mitochondrial DNA damage.104 The discrepancy in whether IR with subsequent MPT formation results in mitophagy may be due to differences in the models used, such as in vitro versus in vivo settings. This discrepancy along with the fact that the molecular constituents of the MPT pores have not been discovered, seek to highlight the need for much more research in this field to better characterize the main players and their interrelationships in MPT pore formation and control of conductance.
CONCLUDING REMARK AND FUTURE DIRECTIONS
Despite 2 decades of research into the pathophysiological basis of liver IR, many questions remain unanswered. The complex interactions woven by each of the cellular contributors is only just beginning to be mapped. The function of different toll-like receptors and their respective DAMP is a relatively new field of research about which little is known. The detrimental means by which disturbances of the homeostatic concentrations of Ca2+, Na+, and H+ arise, and the pathways mediating the consequences of these disturbances are incompletely understood. The structure and regulation of conductance of MPT pores in liver IR injury is poorly characterized and warrants further research. Only by understanding the action of these factors and how they interact with each other will it be possible to design therapeutic strategies to ameliorate liver IR injury.