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Liver transplantation has become the only curative therapy for many patients with end-stage liver disease and/or acute liver failure. Recently, because of the shortage of donor organs, partial orthotopic liver transplantation (OLT) has been developed, and it involves splitting a deceased donor graft or using a partial liver graft from a living donor.1, 2 However, small-for-size liver graft injuries present major obstacles. Small-for-size liver graft injuries are associated not only with high mortality and postoperative complications but also with acute rejection.3–5 Excessive portal inflow induces mechanical injury to small-for-size liver grafts and accelerates hepatic ischemia-reperfusion injury (IRI), especially hepatic microcirculatory disturbances via irreversible sinusoidal endothelial cell (SEC) injury after reperfusion.6, 7
Various therapeutic strategies to protect small-for-size liver grafts, including surgical innovations,8, 9 the use of pharmacological agents,10, 11 and gene therapies,12, 13 have been reported in animal models, but few of these strategies have been clinically applied. Moreover, only a few studies have focused on the nature of preservation solutions.14, 15 Recently, one of the most important advances in liver transplantation has been the development of novel preservation solutions with an extended safe storage time and reduced graft damage. Common preservation solutions, including University of Wisconsin solution, histidine-tryptophan-ketoglutarate (HTK) solution, and Celsior solution, contain agents for osmotic adjustment, antioxidant substances, and energy substrates but no anti-inflammatory agent.16 Therefore, it is hypothesized that adding anti-inflammatory agents with anticoagulant effects such as activated protein C (APC) to preservation solutions might become a novel therapeutic strategy to protect small-for-size liver grafts.
Although APC has been known to be a major physiological anticoagulant,17, 18 recent studies have revealed that APC inhibits proinflammatory cytokine production through the regulation of its transcription, thereby reducing endothelial cell damage through the inactivation of neutrophils.19, 20 Moreover, it has been reported that APC exerts cytoprotective activities independent of APC's anticoagulant activities, which are mediated by endothelial protein C receptor (EPCR)–dependent protease-activated receptor 1 (PAR-1) activation.21 Clinically, the Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study group has reported that treatment with recombinant human APC significantly reduces mortality in patients with severe sepsis, but it may be associated with increased bleeding risks. Therefore, conditions that increase bleeding risks, including recent trauma or surgery (within 12 hours), are considered exclusion criteria for safety reasons.22
The cytoprotective effects of APC have been investigated in IRI models of many organs.23–26 In a previous study, using a rat hepatic warm IRI model, we revealed that APC improves hepatic microcirculation by decreasing the expression of adhesion molecules and preventing leukocyte activation. However, multiple injections of APC are necessary to maintain its cytoprotective action over the long term.27 Consequently, to the best of our knowledge, there have been no data on the impact of APC in liver transplantation. The intravenous administration of APC in major hepatectomy or liver transplantation may be associated with increased risks of perioperative bleeding. However, in liver transplantation, it is hypothesized that the addition of APC to the preservation solution is a simple and promising strategy for alleviating graft injury and prolonging cytoprotective function. Accordingly, the present study was designed to evaluate the cytoprotective effects of a preservation solution containing APC on small-for-size liver grafts through the investigation of the survival, liver function, acute inflammatory response, vascular tone, and apoptosis.
Male Wistar Hannover rats (CLEA Japan, Tokyo, Japan), weighing 190 to 240 g, were used for all experiments. The rats had free access to rat chow and water before the surgical procedures and were kept under constant environmental conditions with a 12-hour light-dark cycle. All experiments complied with the guidelines for animal experiments of the National Institutes of Health (publication 86-23, revised in 1985).
HTK solution (Kohler Chemie, Alsbach, Germany) was used for rinsing and preserving grafts at 2 to 4°C. Human plasma–derived APC was kindly provided by the Chemo-Sero-Therapeutic Research Institute (Kumamoto, Japan) and was directly added to the HTK solution.
Model of Small-for-Size Liver Transplantation
Rats were anesthetized with isoflurane, and the livers were exposed with transversal laparotomy on an isothermal pad (Braintree Scientific, Inc., Braintree, MA) for the maintenance of the body temperature near 37°C during the operation. The OLT method was a modified version of the technique described by Kamada and Calne.28 After systemic heparinization, the donor livers (20% partial liver) were harvested with the modified technique described by Tanaka et al.,29 were flushed through the portal vein, and were stored in HTK solution alone or in HTK solution containing APC. After the removal of the livers from the recipients, the partial liver grafts were anastomosed to the suprahepatic vena cava with 8-0 continuous sutures. The portal vein and infrahepatic vena cava were anastomosed with the cuff technique. The bile duct was anastomosed with an intraluminal stent. The transplantation procedure lasted less than 50 minutes, during which time the portal vein was clamped for 11 to 15 minutes.
To determine the preservation time of the graft, recipient animals were randomly assigned to the following 2 major groups according to the nature of the preservation solution: in the control groups (n = 15), the grafts were flushed and stored in HTK solution alone for 2, 6, or 20 hours; in the APC-treated groups (n = 20), the grafts were flushed and stored in HTK solution containing 100 nM APC for 2, 6, or 20 hours. Five rats in each of the control and APC-treated groups were used for the survival study. Rats that had lived more than 7 days after partial OLT were considered survivors. The 7-day animal survival rates for 2, 6, and 20 hours in the control group versus the APC group were 100% (5/5) versus 100% (5/5), 60% (3/5) versus 100% (5/5), and 0% (0/5) versus 0% (0/5), respectively. On the basis of these results, the preservation time was determined to be 6 hours.
Next, to examine the effectiveness of different APC concentrations in the preservation solution for 6 hours, the APC-treated groups were randomly assigned to 2 different APC concentrations: 10 and 100 nM. The 7-day animal survival rates with 10 and 100 nM APC for 6 hours were 80% (4/5) and 100% (5/5), respectively. Accordingly, the APC concentration was determined to be 100 nM.
According to the results of our preliminary study, the experimental animals were randomly assigned to the following 2 groups: in the control group (n = 30), the grafts were flushed and stored in HTK solution alone for 6 hours, and in the APC-treated group (n = 28), the grafts were flushed and stored in HTK solution containing 100 nM APC for 6 hours.
Ten rats in the control group and 10 rats in the APC-treated group were used for the survival study. Rats that had lived more than 7 days after transplantation were considered survivors.
Liver tissues and blood were sampled 6 and 24 hours after OLT for hepatic protein detection, morphological examination, and liver function tests. Five rats were included at each time point for the control and APC-treated groups. Because 2 rats in the control group died within 24 hours after OLT, 2 rats were added to the control group 24 hours after OLT.
Measurement of the Portal Pressure and Hepatic Microcirculation
Four rats in the control group and 4 rats in the APC-treated group were used for the measurement of the portal pressure and hepatic microcirculation. The ileocolic vein was cannulated by a catheter (24G; Terumo, Tokyo, Japan) to measure the mean portal pressure as described previously.7 This catheter was connected via a pressure transducer (BSM-3201, Nihonkoden, Tokyo, Japan). Hepatic microcirculation was measured with a laser Doppler flowmeter (BRL-100; Bio Research Center, Nagoya, Japan) for 60 minutes of reperfusion as described previously.15 The results were expressed as percentages of the predonation level (whole size).
Measurement of the Serum Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) Levels
Liver injury was quantified by the measurement of the serum ALT and AST levels with a Wako test for aminotransferase (Wako, Osaka, Japan) according to the manufacturer's instructions.
Myeloperoxidase (MPO) Assay
The activity of MPO, an enzyme specific to polymorphonuclear neutrophils and macrophages/monocytes, was used as an index of hepatic leukocyte accumulation.30, 31 Frozen tissues were homogenized in iced 0.5% hexadecyltrimethyl ammonium bromide (Sigma-Aldrich, St. Louis, MO) and 50 mmol of a potassium phosphate buffer solution (pH 5; Sigma-Aldrich). After centrifugation, the supernatants were mixed with a solution of hydrogen peroxide sodium acetate and tetramethyl benzidine (Sigma-Aldrich). The change in absorbance was measured spectrophotometrically at 655 nm. One unit of MPO activity was defined as the quantity of enzyme degrading 1 μmol of peroxide per minute per gram of tissue at 25°C.
Measurement of the Hepatic Tumor Necrosis Factor α (TNF-α) and Interleukin 6 (IL-6) Protein Levels
Liver samples were homogenized in an extraction buffer (50 mmol/L trishydroxymethylaminomethane, pH 7.2; 150 mmol/L NaCl; and Triton X-100) and a protease inhibitor cocktail. The homogenates were shaken on ice for 90 minutes and centrifuged at 3000g and 4°C for 15 minutes. The hepatic levels of TNF-α and IL-6 were determined with commercially available enzyme-linked immunosorbent assays (BD Biosciences, San Diego, CA) according to the manufacturer's instructions.
Measurement of the Serum Hyaluronic Acid Levels
Serum hyaluronic acid levels were measured as a sinusoidal cell function with an assay kit (Fujirebio, Tokyo, Japan) according to the manufacturer's instructions.
Liver specimens were fixed in a 10% buffered formalin solution and embedded in paraffin. Sections (thickness = 5 μm) were then prepared and stained with hematoxylin and eosin. The histological severity of hepatic IRI was graded with modified Suzuki criteria.32 In this classification, sinusoidal congestion, hepatocyte necrosis, and ballooning degeneration were graded from 0 to 4. No necrosis, congestion, or centrilobular ballooning was given a score of 0, whereas severe congestion, ballooning degeneration, and 60% lobular necrosis were given a value of 4.
Paraffin-embedded tissue sections were deparaffinized and rehydrated, and this was followed by a proteinase K treatment (Chemicon, Temecula, CA). Next, sections were incubated in 0.3% H2O2 in methanol to block endogenous peroxidase activity. A primary antibody against macrophage/monocyte (ED-1; Chemicon) was added at an optimal dilution. The secondary antibody and ABC reagents were applied according to the manufacturer's instructions (Vectastain ABC kit, Vector, Burlingame, CA). Color development was induced by incubation with a 3,3-diaminobenzidine substrate (Vector). The neutrophils were stained with a naphthol AS-D chloroacetate esterase staining kit (Sigma-Aldrich) according to the manufacturer's instructions. The results were evaluated via the counting of labeled cells in triplicate with 20 high-power fields (200×) per section.
Liver proteins were extracted with a mammalian cell lysis buffer kit (Sigma-Aldrich). Briefly, liver homogenates were centrifuged at 15,000g for 10 minutes at 4°C. Proteins (50 μg) were separated by 10% to 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (ATTO, Tokyo, Japan) and transferred to a polyvinylidene fluoride membrane (ATTO). The membrane was blocked with 5% dry milk and 0.1% (vol/vol) Tween 20 in phosphate-buffered saline (PBS-T). Blots were incubated with primary antibodies overnight at 4°C. Horseradish peroxidase–conjugated secondary antibodies (Vector) were incubated for 1 hour at room temperature. The immunoreactive signals were visualized with ECL Plus Western blotting detection reagents (Amersham, Little Chalfont, United Kingdom), quantified by scanning densitometry, and presented in comparison with β-actin expression. Appropriate primary antibodies against inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS; BD Biosciences), phosphorylated endothelial nitric oxide synthase (p-eNOS; Cell Signaling, Beverly, MA), and β-actin (Sigma-Aldrich) were used.
Measurement of the Hepatic Protein and Serum Nitric Oxide (NO) Levels
Serum and hepatic NO (nitrite and nitrate) levels were measured with an assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions. Briefly, frozen tissue specimens were homogenized in PBS-T and centrifuged at 10,000g for 20 minutes. After the precipitated samples were passed through a 0.22-μm membrane filter (Millipore, Tokyo, Japan), they were further concentrated with a 30-kDa molecular weight cutoff filter (Millipore) and used for subsequent measurements.
Measurement of the Hepatic Protein and Serum Endothelin 1 (ET-1) Levels
Serum and hepatic ET-1 levels were measured with an assay kit (Cayman Chemical) according to the manufacturer's instructions. Briefly, frozen tissue specimens were homogenized in 6% acetic acid and immediately boiled for 10 minutes to inactivate proteases.33 The extraction of the samples was performed with solid-phase extraction cartridges (Cayman Chemical). ET-1 was eluted with 4% acetic acid in 86% ethanol. The cartridge eluates were vacuum-evaporated, and dried residues were reconstituted with an enzyme immunoassay buffer for subsequent enzyme immunoassay measurements.
Measurement of the Caspase-8 and Caspase-3 Activities
Caspase-8 and caspase-3 activities were measured with an assay kit (Clontech Laboratories, Mountain View, CA) according to the manufacturer's instructions.
Detection of Apoptosis
Apoptosis was detected with the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method. Paraffin-embedded tissue sections were deparaffinized, and the dehydrated sections were treated with the In Situ Cell Death Detection Kit, POD (Roche Diagnostics, Temecula, CA), according to the manufacturer's instructions. The results were scored semiquantitatively via the averaging of the number of TUNEL+ cells per field with 6 fields per tissue sample.
Data were expressed as medians and ranges (minimum to maximum). Differences between the distributions of the APC-treated and control groups were determined with the Mann-Whitney U test for unpaired comparison. For the survival study, a Kaplan-Meier log-rank analysis was performed. All differences were considered statistically significant for P values < 0.05.
APC Prolongs Partial OLT Survival in Small-for-Size Liver Grafts
The APC-treated group showed significantly improved 7-day animal survival (from 60% to 100%) in comparison with the control group (P = 0.021; Fig. 1). Additionally, there was no postoperative bleeding in either group.
APC Improves the Hepatic Microcirculation and Decreases the Initial Portal Pressure
The hepatic microcirculation was significantly improved in the APC-treated group after reperfusion in comparison with the control group (P < 0.05; Fig. 2A). The portal pressure significantly decreased in the APC-treated group between 10 and 20 minutes after reperfusion (P < 0.05; Fig. 2B).
APC Improves Hepatic Function and Ameliorates Hepatocellular Injury
Serum levels of AST and ALT were markedly decreased in the APC-treated group compared to the control group 6 and 24 hours after OLT [the control group versus the APC-treated group at 6/24 hours: 2604 (1829–4481)/1780 (1676–2837) versus 1460 (1171–1654)/953 (791–1115) IU/L for AST, P = 0.012/0.012, and 525 (331–745)/414 (302–664) versus 303 (174–371)/181 (125–267) IU/L for ALT, P = 0.036/0.012; Fig. 3A,B]. According to Suzuki's histological classification using hematoxylin and eosin staining (Fig. 3C–F), the control group showed moderate to severe hepatocyte vacuolization with disruption of the lobular architecture and moderate sinusoidal congestion [Suzuki score at 6/24 hours = 4.5 (2–7)/5.5 (5–10)]. In contrast, the APC-treated group showed minimal hepatocyte vacuolization and sinusoidal congestion 6 and 24 hours after OLT [Suzuki score at 6/24 hours = 2 (1-3)/5 (4-6)].
APC Prevents the Infiltration of Neutrophils and Macrophages
The MPO activity of the liver tissues was significantly reduced in the APC-treated group compared to the control group [3.36 (2.41–6.77)/3.07 (2.52–5.72) versus 1.81 (1.79–3.04)/2.07 (0.46–2.23) U/g at 6/24 hours, P = 0.036/0.012; Fig. 4A]. The APC-treated group showed markedly decreased intrahepatic infiltration of naphthol AS-D–positive cells [10.4 (7.4-12.4)/8.2 (6.0–9.0) versus 5.3 (4.4–6.2)/4.0 (3.6–4.4) at 6/24 hours, P = 0.030/0.029; Fig. 4B–F] and ED-1–positive cells [44.8 (34.0–61.5)/27.6 (22.2–39.5) versus 18.5 (13.0–25.5)/20.0 (18.2–20.2) at 6/24 hours, P = 0.030/0.029; Fig. 4G-K] in comparison with the control group.
APC Decreases the Hepatic Protein Expression of TNF-α and IL-6
The APC-treated group showed significantly decreased protein expression of TNF-α [27.4 (23.8–31.0) versus 16.9 (14.2–21.0) pg/mg of protein, P = 0.012; Fig. 5A] and IL–6 [282 (239–335) versus 196 (180–258) pg/mg of protein, P = 0.021; Fig. 5B] in comparison with the control group 6 hours after OLT. However, there were no significant differences in the protein expression of TNF-α or IL-6 between the 2 groups 24 hours after OLT.
APC Reduces Endothelial Cell Injury After OLT
The APC-treated group showed markedly decreased serum hyaluronic acid levels [532 (415-704)/891 (793–1030) versus 184 (158–293)/484 (335–560) pg/mL at 6/24 hours, P = 0.012/0.012; Fig. 6] in comparison with the control group.
APC Up-Regulates Hepatic eNOS and p-eNOS and Down-Regulates Hepatic iNOS
As shown in Fig. 7A,B, the APC-treated group showed increased protein expression of eNOS (P = 0.030/0.030) and p-eNOS (P = 0.060/0.060) and decreased protein expression of iNOS (P = 0.060/0.060) in comparison with the control group 6 and 24 hours after OLT.
APC Increases Hepatic Levels of NO and Decreases Hepatic/Serum Levels of ET-1
The APC-treated group showed significantly increased protein levels of NO [1.91 (1.09–2.03)/0.82 (0.57–0.92) versus 2.98 (2.03–3.50)/1.12 (0.90–2.53) nmol/mg of protein at 6/24 hours, P = 0.021/0.036; Fig. 8A] and decreased protein levels of ET-1 [0.94 (0.73-1.34)/0.98 (0.87–1.16) versus 0.58 (0.10–0.90)/0.61 (0.47–0.90) pg/mg of protein at 6/24 hours, P = 0.036/0.036; Fig. 8B] in comparison with the control group 6 and 24 hours after OLT. As shown in Fig. 8C,D, there was no significant difference in the serum levels of NO between the 2 groups. The APC-treated group showed significantly decreased serum levels of ET-1 [1.39 (1.20–1.72) versus 0.94 (0.90–1.16) pg/mL, P = 0.011] only 6 hours after OLT in comparison with the control group.
APC Promotes Antiapoptotic Actions
Six hours after OLT, liver tissues of the APC-treated group showed significantly decreased caspase-8 activity [0.33 (0.29-0.49) versus 0.28 (0.22–0.29), P = 0.012; Fig. 9A] and caspase-3 activity [0.33 (0.28–0.46) versus 0.20 (0.16–0.20), P = 0.012; Fig. 9B] and fewer TUNEL-positive cells [11.8 (10.0–12.7) versus 4.1 (3.0–4.5), P = 0.029; Fig. 9D-F] in comparison with the control group.
The mechanisms underlying the dysfunction and failure of small-for-size grafts remain unclear. According to many previous studies, the major problems of small-for-size liver graft failure in the early phase after OLT are due to hepatic sinusoidal damage caused by transient portal hypertension from hemodynamic forces, severe inflammatory response triggered by shear stress, subsequent hepatic microcirculatory disorders due to an imbalance of vascular mediators (ET-1/NO), the higher metabolic burden for regeneration, and up-regulation of apoptotic signals.11, 34–36
In the present study, APC treatment significantly increased 7-day animal survival. As shown in Fig. 1, all deceased animals died within 3 days. Additionally, there were no significant differences in liver function at 7 days after OLT in the survivors of the 2 groups (data not shown). We thought that it was very important to prevent hepatic IRI at an early phase after OLT. APC treatment significantly decreased aminotransferase levels and improved histological features of hepatic IRI within 24 hours after OLT, and this in turn improved animal survival. Furthermore, previous studies demonstrated that early graft function after OLT correlates with the initial hepatic microcirculation just after reperfusion.7, 37, 38 For graft survival, it is essential to improve the hepatic microcirculation and decrease the initial portal pressure just after reperfusion. We showed that the addition of APC to a preservation solution was highly effective in preventing the initial hepatic microcirculatory disturbance just after reperfusion.
APC treatment prevented the infiltration of polymorphonuclear neutrophils and macrophage/monocytes and decreased hepatic expression of TNF-α and IL-6. In particular, TNF-α is a central mediator of the hepatic response to IRI. On the other hand, IL-6 is a typical pleiotropic cytokine that acts on various cells. Recently, IL-6 was shown to induce liver regeneration and reduce hepatic IRI. However, the administration of a high dose of IL-6 is necessary to exert its cytoprotective function.15 On the contrary, IL-6 is considered a proinflammatory cytokine representing the severity of hepatic IRI.39, 40 In the present study, APC treatment remarkably decreased TNF-α and IL-6 hepatic expression at 6 hours but not 24 hours after partial OLT. With a small-for-size graft model, their hepatic protein expression rapidly increased after OLT, peaked at about 6 hours, and then decreased slightly.41, 42 Additionally, APC is rapidly cleared within a few hours after its in vivo administration.43 Therefore, we believe that APC was no longer present 24 hours after partial OLT but had worn off. Because APC treatment could suppress the peak levels of TNF-α and IL-6 at an early phase after OLT, its effect might prevent hepatic IRI and improve animal survival.
Small-for-size liver graft injuries are probably related to microcirculatory disorders due to an imbalance of vasoconstricting and vasorelaxing mediators.38 In particular, SECs play important roles in regulating hepatic vascular resistance, and impaired SECs contribute to increased hepatic vascular resistance, leading to high portal pressure.44 NO and ET-1 are key mediators controlling vascular tone.45 In the present study, APC treatment remarkably attenuated SEC damage. Moreover, APC treatment markedly increased hepatic levels of NO and decreased hepatic levels of ET-1. NO can act as a toxic or protective agent, depending on the conditions.46, 47 The present study demonstrated that APC treatment up-regulated hepatic eNOS expression and down-regulated hepatic iNOS expression. Recently, it was suggested that eNOS-derived NO prevents hepatic IRI.48, 49 As for the precise mechanism, an in vitro study showed that APC activated eNOS via phosphatidylinositol 3-kinase–dependent phosphorylation followed by activation of protein kinase G, and this suggests that APC bound to EPCR might activate the endothelial mitogen-activated protein kinase pathway by a mechanism similar to that of vascular endothelial growth factor, supporting the present results.50 On the other hand, the effects of iNOS-derived NO remain controversial in OLT.51, 52 Accordingly, in the present study, the cytoprotective mechanism of APC treatment may be associated with increased hepatic levels of NO via up-regulated eNOS expression together with down-regulated iNOS expression and decreased hepatic levels of ET-1, which in turn improved hepatic microcirculation.
Apoptosis has been identified as a key mechanism in hepatic IRI.53 In the present study, APC treatment significantly attenuated TNF-α hepatic expression and remarkably reduced the activation of caspase-8 and caspase-3, resulting in the decreased number of TUNEL-positive cells, which was consistent with antiapoptotic effects.
Therefore, these results suggest that a preservation solution containing APC is a potential novel and safe product for small-for-size liver transplantation, alleviating graft injury via anti-inflammatory and antiapoptotic effects and vasorelaxing conditions. Additionally, it is thought that APC in preservation solutions may potentially be used not only for small-for-size grafts but also for whole size grafts and potentially other organs. However, the underlying cytoprotective mechanism during preservation and after reperfusion was not explored enough in the present study. Actually, it was not presented when APC exerted its cytoprotective effects. Because we did not flush the liver graft containing APC prior to transplantation, it was speculated that APC, whose enzymatic activity at the preservation temperature of 4°C is very low, might exert its effects at the time of rewarming. To validate this mechanism, further examinations with another APC group, whose grafts are flushed with only HTK solution just before OLT, are necessary. Furthermore, the present study did not examine the precise mechanism by which APC exerts its cytoprotective effects. In the previous in vitro studies, it has been revealed that its cytoprotective mechanisms are mediated by EPCR-dependent PAR-1 activation.21 To prove this mechanism, further examinations are required with PAR-1 and/or EPCR blockades and cell-permeable peptides.54 Recently, APC variants, which still have cytoprotective activity but lack anticoagulant activity, have been in development.21 These APC mutants might shed light on its cytoprotective mechanism.