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A New Chemical Compound, NecroX-7, Acts as a Necrosis Modulator by Inhibiting High-Mobility Group Box 1 Protein Release During Massive Ischemia-Reperfusion Injury
A New Chemical Compound, NecroX-7, Acts as a Necrosis Modulator by Inhibiting High-Mobility Group Box 1 Protein Release During Massive Ischemia-Reperfusion Injury J.H. Leea, K.M. Parka,*, Y.J. Leea, J.H. Kimb, and S.H. Kimc a
Department of Hepatobiliary and Pancreatic Surgery, Asan Medical Center, University of Ulsan, College of Medicine, Seoul, Korea; Department of Pathology, Asan Medical Center, University of Ulsan, College of Medicine, Seoul, Korea; and cLG Life Sciences, Daejeon, Korea b
ABSTRACT Background. Necrotic cell death is common in a wide variety of pathologic conditions, including ischemia-reperfusion (IR) injury. The aim of this study was to develop an IR injuryeinduced hepatic necrosis model in dogs by means of selective left hepatic inflow occlusion and to test the efficacy of a new chemical compound, NecroX-7, against the IR injuryeinduced hepatic damage. Methods. A group of male Beagle dogs received intravenous infusions of either vehicle or different doses of NecroX-7 (1.5, 4.5, or 13 mg/kg) for a 20-minute period before a 90-minute left hepatic inflow occlusion followed by reperfusion. Results. The gross morphology in the NecroX-7etreated groups after occlusion appeared to be less congested and less swollen than that in vehicle-treated control group. Circulating alanine transaminase and aspartate transaminase levels in the control group were elevated during the course of IR, and were effectively blocked in the 4.5 and 13 mg/kg NecroX7etreated groups. The serum levels of high-mobility group box 1 protein showed a peak at 8 hours after occlusion in control group, and this elevation was significantly blunted by 4.5 mg/kg NecroX-7 treatment. Histologic analysis showed a marked ischemia or IR injuryeinduced hepatocytic degenerations, sinusoidal and portal vein congestions, and inflammatory cell infiltrations in the control group, whereas the treatment groups showed significantly diminished histopathology in a dose-dependent manner. Conclusions. These results demonstrated that NecroX-7 attenuated the hepatocyte lethality caused by hepatic IR injury in a large animal setting. We conclude that NecroX-7 may provide a wide variety of therapeutic options for IR injury in human patients.
A
POPTOSIS has been traditionally considered to be the only mode of cell death in ischemia-reperfusion (IR) injury [1]. However, accumulating evidence suggests that several types of cell death exist during IR injury depending on the intensity and cause of injury: programmed cell death (apoptosis), nonprogrammed cell death (necrosis), and programmed nonapoptotic cell death [2]. Thus, elucidation of the different cell death types is important to prevent or treat human diseases caused by IR injury following procedures such as organ transplantation [3e8], organ resection [9e23], and vascular bypass. High-mobility group box 1 0041-1345/16 http://dx.doi.org/10.1016/j.transproceed.2016.09.046
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protein (HMGB1), a nuclear DNAebinding protein released outside of the cell during necrosis as a damageassociated molecular pattern molecule, is a key alarmin that triggers immune responses [24e27]. Extracellular Funding: Foundation for Industry Cooperation, University of Ulsan (grant no 2009-1244). *Address correspondence to Kwang Min Park, Department of Hepatobiliary and Pancreatic Surgery, Asan Medical Center, University of Ulsan, College of Medicine, Seoul, Korea. E-mail: [email protected] ª 2016 Published by Elsevier Inc. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 48, 3406e3414 (2016)
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release of HMGB1 occurs as a the result of cell membrane rupture during late apoptosis and pathologic cell necrosis [28]. Among the nuclear proteins (including heat shock proteins, soluble S100 proteins, and HMGB1), HMGB1 is the most potent, immunologically suitable, and active alarmin [29]. Moreover, intraperitoneal injection of HMGB1 causes 100% death in mice [30]. Modulation of this key proinflammatory molecule may facilitate control of IR injuries and consequently prevent necrotic cell death [31e33] and further organ damage. However, there are no known inhibitors of HMGB1 release during IR injuryeinduced hepatic necrosis at present. A novel synthetic chemical compound, NecroX-7, has been developed as a potent small molecule modulating necrotic cell death, with a chemical formula of C25H32N4O4S2 and molecular weight 516.67. In the present study, we conducted a large-animal experiment to test the clinical applicability of this new compound as a necrosis modulator via inhibition of HMGB1 release during the cell death process. The specific aims of this study were to establish a necrosis-dominant hepatic IR injury model in beagles by means of 90 minutes of selective left hepatic inflow occlusion of the liver, and to examine the protective effects of NecroX-7 on IR injuryeinduced hepatic necrosis in the model before moving to the clinical setting. METHODS Research Design and Animal Husbandry Twenty-seven healthy male beagle dogs weighing 8.5e11.5 kg were selected for the experiment. Animals were fasted for 24 hours to minimize any effects of hepatic glycogen overload, and they were shaved before surgery, followed by random assignment into the control group A (n ¼ 9) and experimental groups B (n ¼ 6), C (n ¼ 6), and D (n ¼ 6). Premedication with 1.5, 4.5, and 13 mg/kg, respectively, of NecroX-7 was administered. The dogs were intubated with 6e7 endotracheal tubes and were administered general anesthesia with induction by zoletil and maintenance with 2.5% enflurane. Cefazolin and ketorolac were used as the prophylactic antibiotic and analgesic, respectively. All animal protocols and experiments were approved by the Institutional Ethical Committee.
Experimental Material The chemical formula and molecular weight of NecroX-7 are C25H32N4O4S2 and 516.67, respectively. The molecular structure is shown in Fig 1.
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Operative Procedures The dogs were placed in a supine position on the operating table, on top of warm blankets, with warm-water bottles as side supports. The left external jugular vein and right long saphenous vein were cannulated with the use of 21-gauge intravenous catheters by means of an open approach. The right internal carotid artery was dissected and cannulated for full monitoring of hemodynamics during the operation. After achieving secure intravenous access, anesthesia was maintained with inhalation of 2.5% enflurane. Crystalloid fluid was administered at a rate of 15 mL/kg/h. The control group (n ¼ 9) received IR injury with no NecroX-7 pretreatment and the intervention groups B, C, and D (n ¼ 6/ group) received different doses of intravenous NecroX-7 (1.5, 4.5, and 13 mg/kg, respectively) over 20 minutes, before 90 minutes of left hepatic inflow occlusioneinduced ischemia. The concurrent dosage and infusion rate were Cmax 12.5 mg/mL at 20 minutes and Css 5 mg/mL, respectively. The dogs were fully monitored during the operative procedure, and blood pressure, heart rate, respiratory rate, and arterial oxygen saturation assessed every 15 minutes. The operation was performed with the use of a reverse T incision. The hepatoduodenal ligament was isolated after dividing the lesser omentum. The gallbladder and segment 4 of liver were identified, and gently retraced upward following the hilar structures. Figure 2A depicts the left and right lobes of the liver, divided by the Cantlie line (drawn between the gallbladder and suprahepatic inferior vena cava), as well as the 3 major hepatic vasculobiliary inflows: right anterior, right posterior, and left glissonian pedicle. Typical hilar anatomy, with the common bile duct, common hepatic artery, and main branch of the portal vein is shown in Fig 2B. In humans, the right pedicle supplies 70% of the liver volume (right lobe), whereas in beagles, the left pedicle supplies 60%-70% of the total liver volume (left lobe), as shown in our previous autopsy studies. The left hepatic inflow was occluded for 90 minutes, and the liver was reperfused by removing the clamp (Fig 2C). Thus, the model involved 90 minutes of selective left hepatic inflow occlusion, allowing the maintenance of splanchnic blood flow through the right portal flow to trigger the necrosis-dominant cell death pathway in IR injury. After the operation, abdomen and skin were closed. The dogs were maintained in a warm and clean environment with ongoing analgesia and provided a soft high-protein diet. The dogs were killed 48 hours after the procedure.
Blood Sampling and Liver Biopsy Blood sampling (0, 1.5, 2.5, 8, 24, and 48 h) and liver biopsy (0, 1.5, 2.5, and 48 h) were performed throughout the procedure at predetermined intervals (Fig 2, bottom). For each sample, clinical analyses for alanine transaminase (ALT; glutamate pyruvate transaminase) and aspartate transaminase (AST; glutamate oxaloacetate transaminase) were performed, and serum HMGB1 was measured with the use of an enzyme-linked immunosorbent assay. Measurement of the circulating NecroX-7 level for the pharmacokinetic profile was performed by means of a liquid chromatography technique.
Histologic Analysis for IR Injury
Fig 1. Core molecular structure of NecroX-7 derivatives.
Liver biopsies were post-fixed, embedded in paraffin after dehydration, sectioned at 4-mm thickness, stained by means of the Mayer hematoxylin-eosin method, and examined under a light microscope at 200 magnification. Hepatic cell derangement was analyzed semiquantitatively. Samples were assessed for hepatocytic degeneration and necrosis, sinusoidal and portal vein congestion, and
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Fig 2. Procedures for hepatic ischemia-reperfusion operation in beagle dogs. (A) Left and right lobes of canine liver, divided by the Cantlie line, also showing the 3 major hepatic vasculobiliary inflows: right anterior, right posterior, and left glissonian pedicle. (B) Typical hilar anatomy, showing the common bile duct, common hepatic artery, and main branch of the portal vein. (C) The left hepatic inflow was occluded for 90 minutes, and the liver was reperfused for 60 minutes by removing the clamp. (Bottom) Blood sampling and liver biopsy were performed throughout the procedure at the indicated times (arrows). inflammatory cell infiltration. Histologic findings were graded on a semiquantitative scale depending on the percentage of positive findings for each section. Values were assigned: 0 (0%, none), 1 (1%-25%, mild), 2 (26%-50%, moderate), or 3 (51%-100%, marked). In areas of inflammatory cell infiltration, lobular area and periportal areas were calculated separately, and the sum of each used for statistical analysis. All samples were randomized and analyzed blindly. For immunohistochemistry analyses, paraffin-embedded liver sections were subjected to staining for HMGB1 and nitrotyrosine (a marker for cellular oxidative stress). A set of liver samples was processed for electron micrography, and the sections were analyzed for hepatic cell morphology.
Mitochondrial Complex I Activity The mitochondrial complex I activities in a set of liver tissues from test groups were analyzed and compared with those of the control group.
Statistical Analyses Results were analyzed with the use of 2-way analysis of variance followed by post hoc comparisons with Fisher protected least significant difference test for multiple comparisons, with the use of the statistical software Statview (version 4.51; Abacus Concepts, Berkeley, California). P values of <.05 were considered to be statistically significant. All data are presented as mean SEM.
RESULTS
The serum ALT and AST levels in control group A were increased significantly after occlusion, reaching peak values at 24 and 8 hours, respectively, compared with before
occlusion (Fig 3A and B). Notably, the levels of these liver enzymes were markedly attenuated after administration of 4.5 and 13 mg/kg NecroX-7 compared with the control. However, the decrease observed with the 1.5 mg/kg dose failed to reach statistical significance. Serum HMGB1 was significantly elevated in the control group at 8 hours after occlusion, which was effectively suppressed with 4.5 mg/kg NecroX-7, resulting in a trend of slow increase (Fig 3C). The circulating levels of NecroX-7 rapidly peaked with different amplitudes, and were excreted within hours in a dose-dependent manner (Fig 3D). After 90 minutes of inflow occlusion and subsequent reperfusion, the gross hepatic morphology of the left lobe in the control group appeared congested and swollen. These symptoms of IR injury were improved with 4.5 mg/kg NecroX-7 treatment (Fig 4A). Histologic analyses at 90 minutes, 150 minutes, and 48 hours after occlusion revealed marked ischemic necrosis in the control group. In contrast, the group treated with 4.5 mg/kg NecroX-7 showed little evidence of IR injury (Fig 4B). Further detailed microscopic analyses disclosed that NecroX-7 clearly reduced hepatocytic degeneration up to 150 minutes after occlusion, as estimated from the degree of hepatocellular vacuolation and swelling (Fig 5A). NecroX-7 administration also led to a decrease in total congestion (sum of sinusoidal and portal vein congestion), inflammatory cell infiltration in portal space, and irreversible hepatocytic necrosis up to 48 hours after occlusion. The protective effects of NecroX-7 were most marked at
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Fig 3. NecroX-7 effectively attenuates ischemia-reperfusion (IR) injuryeinduced circulating alanine transaminase (ALT), aspartate transaminase (AST), and highmobility group box 1 protein (HMGB1) levels in the beagle model. IR control group (n ¼ 9) received IR injury with no NecroX-7. Three different doses of NecroX-7 (1.5, 4.5, and 13 mg/kg) were intravenously administered to treatment groups (n ¼ 6/group) before 90 minutes of occlusion-induced ischemia. The serum (A) ALT and (B) AST levels were measured by means of clinical analyses, (C) serum HMGB1 by means of enzyme-linked immunosorbent assay, and (D) serum NecroX-7 by means of high-performance liquid chromatogrpahy. Results were analyzed by means of 2-way analysis of variance, followed by post hoc comparisons with Fisher protected least significant difference test. Data are presented as mean SEM. *P < .05 vs IR control at the same time point; **P < .05 vs same group at time 0.
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Fig 4. NecroX-7 effectively improves ischemia-reperfusion (IR) injuryeinduced pathologic hepatic morphology in the beagle model. (A) Representative gross hepatic morphology of the (top) IR control and (bottom) 4.5 mg/kg NecroX-7 groups during the course of 90 minutes of inflow occlusioneinduced ischemia and 60 minutes of reperfusion. (B) Representative histologic analyses of liver sections from (top) IR control and (bottom) 4.5 mg/kg NecroX-7 groups during the course of 90 minutes of occlusion, 60 minutes of reperfusion, and 48 hours after occlusion.
150 minutes after occlusion. NecroX-7 induced a significant reduction in individual symptoms of IR injury in a dosedependent manner (Fig 5B), although the prevention of hepatocytic necrosis was significant only at 48 hours. In the majority of cases, 4.5 and 13 mg/kg NecroX-7 doses were both effective, whereas the 1.5 mg/kg dose failed to have a noticeable effect. The immunohistochemistry for HMGB1 revealed lower extracellular excretion in the NecroX-7etreated group. An electron micrograph of liver sections from control group samples at 150 minutes after occlusion disclosed marked mitochondrial congestion and swelling, as well as loss of cellular membrane. In contrast, the group treated with 4.5 mg/kg NecroX-7 displayed normal mitochondrial morphology with clear folds of the inner membrane structure, the cristae (Fig 6A). Consistent with this finding, mitochondrial complex I activities from the liver tissues of
the NecroX-7 groups were recovered and preserved, at least in part, from IR injuryeinduced suppression in a dosedependent manner (Fig 6B). Liver tissues treated with 4.5 and 13 mg/kg NecroX-7 showed biochemical and histologic evidence of IR injury prevention, but not those treated with 1.5 mg/kg NecroX-7. Additionally, the pharmacokinetic profile of NecroX-7 and prevention of ALT, AST, and HMGB1 release were dose dependent. Based on these results, the protective effect of NecroX-7 was dose dependent, and 4.5 mg/kg was the concentration showing full efficacy. DISCUSSION
Accumulating evidence suggests that cell death is divided into several categories dependent on different signals: programmed cell death (apoptosis), programmed
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Fig 5. NecroX-7 effectively ameliorates ischemia-reperfusion (IR) injury-induced (A) microscopic pathologic hepatic damage and (B) hepatocytic necrosis in the beagle model. IR control group (n ¼ 9) received IR injury with no NecroX-7 treatment. NecroX-7 was intravenously administered to treatment groups (n ¼ 6/group) at 3 doses (1.5, 4.5, and 13 mg/kg) before 90 minutes of occlusioninduced ischemia. Biopsy samples of left hepatic lobes were processed, sectioned, hematoxylin and eosin stained, and histologic observations graded on a semiquantitative scale. Total congestion is the sum of sinusoidal and portal vein congestions. All samples were randomized and analyzed blindly. Results were analyzed by means of 2-way analysis of variance followed by post hoc comparisons with Fisher protected least significant difference test. Data from liver biopsy samples at 2.5 hours after initiation of occlusion were collected and processed for separate statistical analyses. Data from the basal (time 0) samples were pooled and plotted as “normal” (n ¼ 27). Data are presented as mean SEM. *P < .05 vs IR control at the same time point.
nonapoptotic cell death (autophagy and necroptosis), and pathologic necrosis [34]. Distinct cell signaling cascades are responsible for the different types of cell death [35e37]. However, a combination of cell death pathways is often involved, depending on the duration and intensity of injury; these combinations can be broadly defined as apoptosis- or necrosis-dominant. Elucidation of the mechanisms underlying these two cell death pathways is important for the prevention and treatment of several human diseases, because necrotic cell death, but not apoptosis, causes inflammation which may be harmful for cells and tissues [38]. We previously reported a pilot study with the use of a 60-minute ischemia model [39]. To achieve the effect of NecroX-7 in enhanced ischemia liver, we selectively occluded left hepatic inflow for 90 minutes, allowing mesenteric blood flow via the right hemiliver. In a recent report, IR injury encountered in multiple clinical settings, such as transplantation, partial hepatectomy, and trauma, is an adaptive inflammatory process caused by pathologic
necrotic cell death or later stages of apoptotic cell death [40]. The interactions between several proinflammatory proteins (heat shock protein, S100, HMGB1) and their pattern recognition receptors (receptor for advanced glycation endproducts [RAGE], Toll-like receptors [TLRs], Syndecan 1) trigger destructive immune responses during massive IR injury via nuclear factor kB activation [41]. HMGB1 is the key alarmin that activates this innate immune response. HMGB1, first identified by Goodwin et al in 1973, is a 25e30 kD DNA-binding protein that binds and bends target DNA and promotes protein assembly on specific DNA sequences [25]. The human HMGB1 gene consists of 6 exons and is located on chromosome 13q12 [42,43]. The versatile HMGB1 protein has been characterized as multifunctional following its initial discovery in the cytoplasm and extracellular area [44e46]. Within the nucleus, HMGB1 stabilizes nucleosomes and regulates the transcription of many genes. On release outside of the cell, HMGB1 binds to cellular receptors with high affinity and masquerades as a
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Fig 6. NecroX-7 effectively normalizes ischemia-reperfusion (IR) injury-einduced pathologic hepatic mitochondrial function in the beagle model. (A) Representative electron micrographs of liver sections from the (left) IR control and (right) 4.5 mg/kg NecroX-7 groups after 90 minutes of inflow occlusioneinduced ischemia and 60 minutes of reperfusion. The bottom panels represent a higher magnification. (B) Mitochondrial complex I activities were measured from a set of liver tissue samples. Pooled basal (time 0) samples were plotted as “normal.”
cytokine to activate endothelial cells, promoting angiogenesis and extravascular emigration of inflammatory and stem cells, and leading to inflammation [47,48]. A number of transmembrane proteins (RAGE, TLR2, TLR4, and Syndecan-1) have been identified as potential cellular receptors. RAGE is activated by a wide range of ligands, including HMGB1 [49,50]. The proinflammatory effects of HMGB1 are mostly mediated via interactions with RAGE [25]. Necrotic cell death was detected based on rapid elevation of HMGB1 in our study model. We further postulated that prevention of HMGB1 release, whether actively secreted by phagocytes during the inflammatory process or passively released during pathologic oncotic necrosis, could represent a novel form of therapy in the field of liver disease caused by severe IR injury [51e55]. To further examine this hypothesis, we examined the effectiveness of a new synthetic chemical compound, NecroX-7, which acts as an inhibitor of necrotic cell death by preventing of HMGB1 release during severe IR injury, in a beagle model. NecroX-7 is a water-soluble small molecule that is easily and rapidly absorbed into the cytoplasm. Preoperative single intravenous injection of NecroX-7 prevented IR injury, as evident from the suppression of HMGB1 release and ALT, as well as lower levels of inflammatory cell recruitment. Furthermore, the duration of action of NecroX-7 was very short and dose dependent, suggesting the possibility of multiple dosing depending on the treatment indication. We proposed that NecroX-7 acts mainly in the mitochondrial matrix, in view of the finding that NecroX-7 protected mitochondrial complex I activity from IR injuryeinduced stress in a dose-dependent manner. However, this hypothesis needs further confirmation. Depending on the availability of mitochondrial adenosine triphosphate (ATP), necrosis with ATP depletion or ATPinduced apoptosis may occur [56,57]. Data from the
present study showed that regardless of the initiation process for necrosis, 4.5 mg/kg NecroX-7 facilitated the conservation of mitochondrial activity by protecting complex I activity. The present study is significant in a number of ways. First, we successfully developed a practical experimental model for IR injuryeinduced necrosis-dominant cell death in beagles for the first time as an essential step to assessing the efficacy of NecroX-7. The beagles in the control as well as the NecroX-7 treatment groups displayed no symptoms of major complications and survived for 3 days until killed for autopsy. Second, a significantly protective effect of NecroX7 against IR injuryeinduced necrosis was evident, in view of reduced HMGB1 release in the treatment groups. The magnitude of differences in ALT and AST levels between the IR control and treatment groups was striking in that the maximum peaks of ALT and AST of the control group were significantly higher than those of the treatment groups. These biochemical results were supported by significantly gross morphologic differences of the liver, in addition to the microscopic observations of liver histology, which showed considerably lower inflammatory cell infiltration in the treatment groups. Our data support multiple potential applications of NecroX-7 in several clinical situations where hepatocyte necrosis is the main cause of morbidity and mortality. NecroX-7 treatment may increase the number of patients who can undergo curative surgical resections for hepatic malignancy on the basis of inadequate future remnant liver volume, especially in cirrhotic liver. This would have a similar effect on liver transplantation, ie, the deceased marginal donor organs may become viable for transplantation, leading to improvements in the number of transplant recipients. Transcatheter arterial chemoembolization (TACE) patients could benefit from the new drug for advanced
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hepatocellular carcinoma (HCC) and nontransplantable recurrent HCC [58]. TACE for HCC treatment uses a combination of direct delivery of chemotherapeutic agents to the tumor and embolization of the hepatic artery by means of a percutaneous method. Although TACE is an effective mode of treatment for a number of patients, it may precipitate liver failure by damaging the surrounding nontumorous healthy hepatocytes. NecroX-7 may be applied to protect normal hepatocytes around the tumor, thus preventing liver injury. Furthermore, in view of the correlation between HMGB1 and adverse effects on tumor invasion and metastasis, it is proposed that NecroX-7 ameliorates the malignant potentials of hepatobiliary and pancreas cancers, which is a further focus of research. In conclusion, we demonstrated that the cell death pathway in massive IR injury is necrosis dominant and that the novel compound, NecroX-7, prevents hepatic IR injuryeinduced lethality, even at concentrations as low as 4.5 mg/kg, via reduction of HMGB1 release in a large animal setting. Thus, NecroX-7 may present an effective therapeutic option in a wide variety of human disorders, particularly in hepatobiliary diseases. The current findings provide a platform for advancing NecroX-7 to clinical trials for several human disease caused by IR injury. REFERENCES [1] Bilbao G, Contreras JL, Eckhoff DE, et al. Reduction of ischemia-reperfusion injury of the liver by in vivo adenovirusmediated gene transfer of the antiapoptotic Bcl-2 gene. Ann Surg 1999;230:185e93. [2] Degterev A, Yuan J. Expansion and evolution of cell death programmes. Nat Rev Mol Cell Biol 2008;9:378e90. [3] Clavien PA, Harvey PR, Strasberg SM. Preservation and reperfusion injuries in liver allografts. An overview and synthesis of current studies. Transplantation 1992;53:957e78. [4] Riordan S, Williams R. Bioartificial liver support: developments in hepatocyte culture and bioreactor design. Br Med Bull 1997;53:730e44. [5] Broelsch CE, Testa G, Alexandrou A, et al. Living related liver transplantation: medical and social aspects of a controversial therapy. Gut 2002;50:143e5. [6] Naruse K. Artificial liver support: future aspects. J Artif Organs 2005;8:71e6. [7] Naruse K, Tang W, Makuuch M. Artificial and bioartificial liver support: a review of perfusion treatment for hepatic failure patients. World J Gastroenterol 2007;13:1516e21. [8] Yu CB, Pan XP, Li LJ. Progress in bioreactors of bioartificial livers. Hepatobiliary Pancreat Dis Int 2009;8:134e40. [9] Delva E, Camus Y, Nordlinger B, et al. Vascular occlusions for liver resections. Operative management and tolerance to hepatic ischemia: 142 cases. Ann Surg 1989;209:211e8. [10] Elias D, Desruennes E, Lasser P. Prolonged intermittent clamping of the portal triad during hepatectomy. Br J Surg 1991;78: 42e4. [11] Hannoun L, Borie D, Delva E, et al. Liver resection with normothermic ischaemia exceeding 1 h. Br J Surg 1993;80:1161e5. [12] Belghiti J, Noun R, Malafosse R, et al. Continuous versus intermittent portal triad clamping for liver resection: a controlled study. Ann Surg 1999;229:369e75. [13] Man K, Fan ST, Ng IO, et al. Tolerance of the liver to intermittent pringle maneuver in hepatectomy for liver tumors. Arch Surg 1999;134:533e9.
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LEE, PARK, LEE ET AL [48] Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 2002;418:191e5. [49] Fink MP. Bench-to-bedside review: High-mobility group box 1 and critical illness. Crit Care 2007;11:229. [50] Andersson U, Erlandsson-Harris H. HMGB1 is a potent trigger of arthritis. J Intern Med 2004;255:344e50. [51] Wang H, Zhu S, Zhou R, et al. Therapeutic potential of HMGB1-targeting agents in sepsis. Expert Rev Mol Med 2008;10:e32. [52] Ferrari FS, Stella A, Pasquinucci P, et al. Treatment of small hepatocellular carcinoma: a comparison of techniques and longterm results. Eur J Gastroenterol Hepatol 2006;18:659e72. [53] Arase Y, Ikeda K, Murashima N, et al. The long term efficacy of glycyrrhizin in chronic hepatitis C patients. Cancer 1997;79:1494e500. [54] Taguchi A, Blood DC, del Toro G, et al. Blockade of RAGE-amphoterin signalling suppresses tumour growth and metastases. Nature 2000;405:354e60. [55] Yasuda T, Ueda T, Takeyama Y, et al. Significant increase of serum high-mobility group box chromosomal protein 1 levels in patients with severe acute pancreatitis. Pancreas 2006;33: 359e63. [56] Lemasters JJ. Necrapoptosis and the mitochondrial permeability transition: shared pathways to necrosis and apoptosis. Am J Physiol 1999;276:G1e6. [57] Lemasters JJ. Modulation of mitochondrial membrane permeability in pathogenesis, autophagy and control of metabolism. J Gastroenterol Hepatol 2007;22(Suppl 1):S31e7. [58] Maleux G, van Malenstein H, Vandecaveye V, et al. Transcatheter chemoembolization of unresectable hepatocellular carcinoma: current knowledge and future directions. Dig Dis 2009;27:157e63.
Department of Hepatobiliary and Pancreatic Surgery, Asan Medical Center, University of Ulsan, College of Medicine, Seoul, Korea; Department of Pathology, Asan Medical Center, University of Ulsan, College of Medicine, Seoul, Korea; and cLG Life Sciences, Daejeon, Korea b
ABSTRACT Background. Necrotic cell death is common in a wide variety of pathologic conditions, including ischemia-reperfusion (IR) injury. The aim of this study was to develop an IR injuryeinduced hepatic necrosis model in dogs by means of selective left hepatic inflow occlusion and to test the efficacy of a new chemical compound, NecroX-7, against the IR injuryeinduced hepatic damage. Methods. A group of male Beagle dogs received intravenous infusions of either vehicle or different doses of NecroX-7 (1.5, 4.5, or 13 mg/kg) for a 20-minute period before a 90-minute left hepatic inflow occlusion followed by reperfusion. Results. The gross morphology in the NecroX-7etreated groups after occlusion appeared to be less congested and less swollen than that in vehicle-treated control group. Circulating alanine transaminase and aspartate transaminase levels in the control group were elevated during the course of IR, and were effectively blocked in the 4.5 and 13 mg/kg NecroX7etreated groups. The serum levels of high-mobility group box 1 protein showed a peak at 8 hours after occlusion in control group, and this elevation was significantly blunted by 4.5 mg/kg NecroX-7 treatment. Histologic analysis showed a marked ischemia or IR injuryeinduced hepatocytic degenerations, sinusoidal and portal vein congestions, and inflammatory cell infiltrations in the control group, whereas the treatment groups showed significantly diminished histopathology in a dose-dependent manner. Conclusions. These results demonstrated that NecroX-7 attenuated the hepatocyte lethality caused by hepatic IR injury in a large animal setting. We conclude that NecroX-7 may provide a wide variety of therapeutic options for IR injury in human patients.
A
POPTOSIS has been traditionally considered to be the only mode of cell death in ischemia-reperfusion (IR) injury [1]. However, accumulating evidence suggests that several types of cell death exist during IR injury depending on the intensity and cause of injury: programmed cell death (apoptosis), nonprogrammed cell death (necrosis), and programmed nonapoptotic cell death [2]. Thus, elucidation of the different cell death types is important to prevent or treat human diseases caused by IR injury following procedures such as organ transplantation [3e8], organ resection [9e23], and vascular bypass. High-mobility group box 1 0041-1345/16 http://dx.doi.org/10.1016/j.transproceed.2016.09.046
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protein (HMGB1), a nuclear DNAebinding protein released outside of the cell during necrosis as a damageassociated molecular pattern molecule, is a key alarmin that triggers immune responses [24e27]. Extracellular Funding: Foundation for Industry Cooperation, University of Ulsan (grant no 2009-1244). *Address correspondence to Kwang Min Park, Department of Hepatobiliary and Pancreatic Surgery, Asan Medical Center, University of Ulsan, College of Medicine, Seoul, Korea. E-mail: [email protected] ª 2016 Published by Elsevier Inc. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 48, 3406e3414 (2016)
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release of HMGB1 occurs as a the result of cell membrane rupture during late apoptosis and pathologic cell necrosis [28]. Among the nuclear proteins (including heat shock proteins, soluble S100 proteins, and HMGB1), HMGB1 is the most potent, immunologically suitable, and active alarmin [29]. Moreover, intraperitoneal injection of HMGB1 causes 100% death in mice [30]. Modulation of this key proinflammatory molecule may facilitate control of IR injuries and consequently prevent necrotic cell death [31e33] and further organ damage. However, there are no known inhibitors of HMGB1 release during IR injuryeinduced hepatic necrosis at present. A novel synthetic chemical compound, NecroX-7, has been developed as a potent small molecule modulating necrotic cell death, with a chemical formula of C25H32N4O4S2 and molecular weight 516.67. In the present study, we conducted a large-animal experiment to test the clinical applicability of this new compound as a necrosis modulator via inhibition of HMGB1 release during the cell death process. The specific aims of this study were to establish a necrosis-dominant hepatic IR injury model in beagles by means of 90 minutes of selective left hepatic inflow occlusion of the liver, and to examine the protective effects of NecroX-7 on IR injuryeinduced hepatic necrosis in the model before moving to the clinical setting. METHODS Research Design and Animal Husbandry Twenty-seven healthy male beagle dogs weighing 8.5e11.5 kg were selected for the experiment. Animals were fasted for 24 hours to minimize any effects of hepatic glycogen overload, and they were shaved before surgery, followed by random assignment into the control group A (n ¼ 9) and experimental groups B (n ¼ 6), C (n ¼ 6), and D (n ¼ 6). Premedication with 1.5, 4.5, and 13 mg/kg, respectively, of NecroX-7 was administered. The dogs were intubated with 6e7 endotracheal tubes and were administered general anesthesia with induction by zoletil and maintenance with 2.5% enflurane. Cefazolin and ketorolac were used as the prophylactic antibiotic and analgesic, respectively. All animal protocols and experiments were approved by the Institutional Ethical Committee.
Experimental Material The chemical formula and molecular weight of NecroX-7 are C25H32N4O4S2 and 516.67, respectively. The molecular structure is shown in Fig 1.
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Operative Procedures The dogs were placed in a supine position on the operating table, on top of warm blankets, with warm-water bottles as side supports. The left external jugular vein and right long saphenous vein were cannulated with the use of 21-gauge intravenous catheters by means of an open approach. The right internal carotid artery was dissected and cannulated for full monitoring of hemodynamics during the operation. After achieving secure intravenous access, anesthesia was maintained with inhalation of 2.5% enflurane. Crystalloid fluid was administered at a rate of 15 mL/kg/h. The control group (n ¼ 9) received IR injury with no NecroX-7 pretreatment and the intervention groups B, C, and D (n ¼ 6/ group) received different doses of intravenous NecroX-7 (1.5, 4.5, and 13 mg/kg, respectively) over 20 minutes, before 90 minutes of left hepatic inflow occlusioneinduced ischemia. The concurrent dosage and infusion rate were Cmax 12.5 mg/mL at 20 minutes and Css 5 mg/mL, respectively. The dogs were fully monitored during the operative procedure, and blood pressure, heart rate, respiratory rate, and arterial oxygen saturation assessed every 15 minutes. The operation was performed with the use of a reverse T incision. The hepatoduodenal ligament was isolated after dividing the lesser omentum. The gallbladder and segment 4 of liver were identified, and gently retraced upward following the hilar structures. Figure 2A depicts the left and right lobes of the liver, divided by the Cantlie line (drawn between the gallbladder and suprahepatic inferior vena cava), as well as the 3 major hepatic vasculobiliary inflows: right anterior, right posterior, and left glissonian pedicle. Typical hilar anatomy, with the common bile duct, common hepatic artery, and main branch of the portal vein is shown in Fig 2B. In humans, the right pedicle supplies 70% of the liver volume (right lobe), whereas in beagles, the left pedicle supplies 60%-70% of the total liver volume (left lobe), as shown in our previous autopsy studies. The left hepatic inflow was occluded for 90 minutes, and the liver was reperfused by removing the clamp (Fig 2C). Thus, the model involved 90 minutes of selective left hepatic inflow occlusion, allowing the maintenance of splanchnic blood flow through the right portal flow to trigger the necrosis-dominant cell death pathway in IR injury. After the operation, abdomen and skin were closed. The dogs were maintained in a warm and clean environment with ongoing analgesia and provided a soft high-protein diet. The dogs were killed 48 hours after the procedure.
Blood Sampling and Liver Biopsy Blood sampling (0, 1.5, 2.5, 8, 24, and 48 h) and liver biopsy (0, 1.5, 2.5, and 48 h) were performed throughout the procedure at predetermined intervals (Fig 2, bottom). For each sample, clinical analyses for alanine transaminase (ALT; glutamate pyruvate transaminase) and aspartate transaminase (AST; glutamate oxaloacetate transaminase) were performed, and serum HMGB1 was measured with the use of an enzyme-linked immunosorbent assay. Measurement of the circulating NecroX-7 level for the pharmacokinetic profile was performed by means of a liquid chromatography technique.
Histologic Analysis for IR Injury
Fig 1. Core molecular structure of NecroX-7 derivatives.
Liver biopsies were post-fixed, embedded in paraffin after dehydration, sectioned at 4-mm thickness, stained by means of the Mayer hematoxylin-eosin method, and examined under a light microscope at 200 magnification. Hepatic cell derangement was analyzed semiquantitatively. Samples were assessed for hepatocytic degeneration and necrosis, sinusoidal and portal vein congestion, and
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Fig 2. Procedures for hepatic ischemia-reperfusion operation in beagle dogs. (A) Left and right lobes of canine liver, divided by the Cantlie line, also showing the 3 major hepatic vasculobiliary inflows: right anterior, right posterior, and left glissonian pedicle. (B) Typical hilar anatomy, showing the common bile duct, common hepatic artery, and main branch of the portal vein. (C) The left hepatic inflow was occluded for 90 minutes, and the liver was reperfused for 60 minutes by removing the clamp. (Bottom) Blood sampling and liver biopsy were performed throughout the procedure at the indicated times (arrows). inflammatory cell infiltration. Histologic findings were graded on a semiquantitative scale depending on the percentage of positive findings for each section. Values were assigned: 0 (0%, none), 1 (1%-25%, mild), 2 (26%-50%, moderate), or 3 (51%-100%, marked). In areas of inflammatory cell infiltration, lobular area and periportal areas were calculated separately, and the sum of each used for statistical analysis. All samples were randomized and analyzed blindly. For immunohistochemistry analyses, paraffin-embedded liver sections were subjected to staining for HMGB1 and nitrotyrosine (a marker for cellular oxidative stress). A set of liver samples was processed for electron micrography, and the sections were analyzed for hepatic cell morphology.
Mitochondrial Complex I Activity The mitochondrial complex I activities in a set of liver tissues from test groups were analyzed and compared with those of the control group.
Statistical Analyses Results were analyzed with the use of 2-way analysis of variance followed by post hoc comparisons with Fisher protected least significant difference test for multiple comparisons, with the use of the statistical software Statview (version 4.51; Abacus Concepts, Berkeley, California). P values of <.05 were considered to be statistically significant. All data are presented as mean SEM.
RESULTS
The serum ALT and AST levels in control group A were increased significantly after occlusion, reaching peak values at 24 and 8 hours, respectively, compared with before
occlusion (Fig 3A and B). Notably, the levels of these liver enzymes were markedly attenuated after administration of 4.5 and 13 mg/kg NecroX-7 compared with the control. However, the decrease observed with the 1.5 mg/kg dose failed to reach statistical significance. Serum HMGB1 was significantly elevated in the control group at 8 hours after occlusion, which was effectively suppressed with 4.5 mg/kg NecroX-7, resulting in a trend of slow increase (Fig 3C). The circulating levels of NecroX-7 rapidly peaked with different amplitudes, and were excreted within hours in a dose-dependent manner (Fig 3D). After 90 minutes of inflow occlusion and subsequent reperfusion, the gross hepatic morphology of the left lobe in the control group appeared congested and swollen. These symptoms of IR injury were improved with 4.5 mg/kg NecroX-7 treatment (Fig 4A). Histologic analyses at 90 minutes, 150 minutes, and 48 hours after occlusion revealed marked ischemic necrosis in the control group. In contrast, the group treated with 4.5 mg/kg NecroX-7 showed little evidence of IR injury (Fig 4B). Further detailed microscopic analyses disclosed that NecroX-7 clearly reduced hepatocytic degeneration up to 150 minutes after occlusion, as estimated from the degree of hepatocellular vacuolation and swelling (Fig 5A). NecroX-7 administration also led to a decrease in total congestion (sum of sinusoidal and portal vein congestion), inflammatory cell infiltration in portal space, and irreversible hepatocytic necrosis up to 48 hours after occlusion. The protective effects of NecroX-7 were most marked at
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Fig 3. NecroX-7 effectively attenuates ischemia-reperfusion (IR) injuryeinduced circulating alanine transaminase (ALT), aspartate transaminase (AST), and highmobility group box 1 protein (HMGB1) levels in the beagle model. IR control group (n ¼ 9) received IR injury with no NecroX-7. Three different doses of NecroX-7 (1.5, 4.5, and 13 mg/kg) were intravenously administered to treatment groups (n ¼ 6/group) before 90 minutes of occlusion-induced ischemia. The serum (A) ALT and (B) AST levels were measured by means of clinical analyses, (C) serum HMGB1 by means of enzyme-linked immunosorbent assay, and (D) serum NecroX-7 by means of high-performance liquid chromatogrpahy. Results were analyzed by means of 2-way analysis of variance, followed by post hoc comparisons with Fisher protected least significant difference test. Data are presented as mean SEM. *P < .05 vs IR control at the same time point; **P < .05 vs same group at time 0.
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Fig 4. NecroX-7 effectively improves ischemia-reperfusion (IR) injuryeinduced pathologic hepatic morphology in the beagle model. (A) Representative gross hepatic morphology of the (top) IR control and (bottom) 4.5 mg/kg NecroX-7 groups during the course of 90 minutes of inflow occlusioneinduced ischemia and 60 minutes of reperfusion. (B) Representative histologic analyses of liver sections from (top) IR control and (bottom) 4.5 mg/kg NecroX-7 groups during the course of 90 minutes of occlusion, 60 minutes of reperfusion, and 48 hours after occlusion.
150 minutes after occlusion. NecroX-7 induced a significant reduction in individual symptoms of IR injury in a dosedependent manner (Fig 5B), although the prevention of hepatocytic necrosis was significant only at 48 hours. In the majority of cases, 4.5 and 13 mg/kg NecroX-7 doses were both effective, whereas the 1.5 mg/kg dose failed to have a noticeable effect. The immunohistochemistry for HMGB1 revealed lower extracellular excretion in the NecroX-7etreated group. An electron micrograph of liver sections from control group samples at 150 minutes after occlusion disclosed marked mitochondrial congestion and swelling, as well as loss of cellular membrane. In contrast, the group treated with 4.5 mg/kg NecroX-7 displayed normal mitochondrial morphology with clear folds of the inner membrane structure, the cristae (Fig 6A). Consistent with this finding, mitochondrial complex I activities from the liver tissues of
the NecroX-7 groups were recovered and preserved, at least in part, from IR injuryeinduced suppression in a dosedependent manner (Fig 6B). Liver tissues treated with 4.5 and 13 mg/kg NecroX-7 showed biochemical and histologic evidence of IR injury prevention, but not those treated with 1.5 mg/kg NecroX-7. Additionally, the pharmacokinetic profile of NecroX-7 and prevention of ALT, AST, and HMGB1 release were dose dependent. Based on these results, the protective effect of NecroX-7 was dose dependent, and 4.5 mg/kg was the concentration showing full efficacy. DISCUSSION
Accumulating evidence suggests that cell death is divided into several categories dependent on different signals: programmed cell death (apoptosis), programmed
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Fig 5. NecroX-7 effectively ameliorates ischemia-reperfusion (IR) injury-induced (A) microscopic pathologic hepatic damage and (B) hepatocytic necrosis in the beagle model. IR control group (n ¼ 9) received IR injury with no NecroX-7 treatment. NecroX-7 was intravenously administered to treatment groups (n ¼ 6/group) at 3 doses (1.5, 4.5, and 13 mg/kg) before 90 minutes of occlusioninduced ischemia. Biopsy samples of left hepatic lobes were processed, sectioned, hematoxylin and eosin stained, and histologic observations graded on a semiquantitative scale. Total congestion is the sum of sinusoidal and portal vein congestions. All samples were randomized and analyzed blindly. Results were analyzed by means of 2-way analysis of variance followed by post hoc comparisons with Fisher protected least significant difference test. Data from liver biopsy samples at 2.5 hours after initiation of occlusion were collected and processed for separate statistical analyses. Data from the basal (time 0) samples were pooled and plotted as “normal” (n ¼ 27). Data are presented as mean SEM. *P < .05 vs IR control at the same time point.
nonapoptotic cell death (autophagy and necroptosis), and pathologic necrosis [34]. Distinct cell signaling cascades are responsible for the different types of cell death [35e37]. However, a combination of cell death pathways is often involved, depending on the duration and intensity of injury; these combinations can be broadly defined as apoptosis- or necrosis-dominant. Elucidation of the mechanisms underlying these two cell death pathways is important for the prevention and treatment of several human diseases, because necrotic cell death, but not apoptosis, causes inflammation which may be harmful for cells and tissues [38]. We previously reported a pilot study with the use of a 60-minute ischemia model [39]. To achieve the effect of NecroX-7 in enhanced ischemia liver, we selectively occluded left hepatic inflow for 90 minutes, allowing mesenteric blood flow via the right hemiliver. In a recent report, IR injury encountered in multiple clinical settings, such as transplantation, partial hepatectomy, and trauma, is an adaptive inflammatory process caused by pathologic
necrotic cell death or later stages of apoptotic cell death [40]. The interactions between several proinflammatory proteins (heat shock protein, S100, HMGB1) and their pattern recognition receptors (receptor for advanced glycation endproducts [RAGE], Toll-like receptors [TLRs], Syndecan 1) trigger destructive immune responses during massive IR injury via nuclear factor kB activation [41]. HMGB1 is the key alarmin that activates this innate immune response. HMGB1, first identified by Goodwin et al in 1973, is a 25e30 kD DNA-binding protein that binds and bends target DNA and promotes protein assembly on specific DNA sequences [25]. The human HMGB1 gene consists of 6 exons and is located on chromosome 13q12 [42,43]. The versatile HMGB1 protein has been characterized as multifunctional following its initial discovery in the cytoplasm and extracellular area [44e46]. Within the nucleus, HMGB1 stabilizes nucleosomes and regulates the transcription of many genes. On release outside of the cell, HMGB1 binds to cellular receptors with high affinity and masquerades as a
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Fig 6. NecroX-7 effectively normalizes ischemia-reperfusion (IR) injury-einduced pathologic hepatic mitochondrial function in the beagle model. (A) Representative electron micrographs of liver sections from the (left) IR control and (right) 4.5 mg/kg NecroX-7 groups after 90 minutes of inflow occlusioneinduced ischemia and 60 minutes of reperfusion. The bottom panels represent a higher magnification. (B) Mitochondrial complex I activities were measured from a set of liver tissue samples. Pooled basal (time 0) samples were plotted as “normal.”
cytokine to activate endothelial cells, promoting angiogenesis and extravascular emigration of inflammatory and stem cells, and leading to inflammation [47,48]. A number of transmembrane proteins (RAGE, TLR2, TLR4, and Syndecan-1) have been identified as potential cellular receptors. RAGE is activated by a wide range of ligands, including HMGB1 [49,50]. The proinflammatory effects of HMGB1 are mostly mediated via interactions with RAGE [25]. Necrotic cell death was detected based on rapid elevation of HMGB1 in our study model. We further postulated that prevention of HMGB1 release, whether actively secreted by phagocytes during the inflammatory process or passively released during pathologic oncotic necrosis, could represent a novel form of therapy in the field of liver disease caused by severe IR injury [51e55]. To further examine this hypothesis, we examined the effectiveness of a new synthetic chemical compound, NecroX-7, which acts as an inhibitor of necrotic cell death by preventing of HMGB1 release during severe IR injury, in a beagle model. NecroX-7 is a water-soluble small molecule that is easily and rapidly absorbed into the cytoplasm. Preoperative single intravenous injection of NecroX-7 prevented IR injury, as evident from the suppression of HMGB1 release and ALT, as well as lower levels of inflammatory cell recruitment. Furthermore, the duration of action of NecroX-7 was very short and dose dependent, suggesting the possibility of multiple dosing depending on the treatment indication. We proposed that NecroX-7 acts mainly in the mitochondrial matrix, in view of the finding that NecroX-7 protected mitochondrial complex I activity from IR injuryeinduced stress in a dose-dependent manner. However, this hypothesis needs further confirmation. Depending on the availability of mitochondrial adenosine triphosphate (ATP), necrosis with ATP depletion or ATPinduced apoptosis may occur [56,57]. Data from the
present study showed that regardless of the initiation process for necrosis, 4.5 mg/kg NecroX-7 facilitated the conservation of mitochondrial activity by protecting complex I activity. The present study is significant in a number of ways. First, we successfully developed a practical experimental model for IR injuryeinduced necrosis-dominant cell death in beagles for the first time as an essential step to assessing the efficacy of NecroX-7. The beagles in the control as well as the NecroX-7 treatment groups displayed no symptoms of major complications and survived for 3 days until killed for autopsy. Second, a significantly protective effect of NecroX7 against IR injuryeinduced necrosis was evident, in view of reduced HMGB1 release in the treatment groups. The magnitude of differences in ALT and AST levels between the IR control and treatment groups was striking in that the maximum peaks of ALT and AST of the control group were significantly higher than those of the treatment groups. These biochemical results were supported by significantly gross morphologic differences of the liver, in addition to the microscopic observations of liver histology, which showed considerably lower inflammatory cell infiltration in the treatment groups. Our data support multiple potential applications of NecroX-7 in several clinical situations where hepatocyte necrosis is the main cause of morbidity and mortality. NecroX-7 treatment may increase the number of patients who can undergo curative surgical resections for hepatic malignancy on the basis of inadequate future remnant liver volume, especially in cirrhotic liver. This would have a similar effect on liver transplantation, ie, the deceased marginal donor organs may become viable for transplantation, leading to improvements in the number of transplant recipients. Transcatheter arterial chemoembolization (TACE) patients could benefit from the new drug for advanced
INHIBITION OF IR INJURYeINDUCED HEPATIC NECROSIS
hepatocellular carcinoma (HCC) and nontransplantable recurrent HCC [58]. TACE for HCC treatment uses a combination of direct delivery of chemotherapeutic agents to the tumor and embolization of the hepatic artery by means of a percutaneous method. Although TACE is an effective mode of treatment for a number of patients, it may precipitate liver failure by damaging the surrounding nontumorous healthy hepatocytes. NecroX-7 may be applied to protect normal hepatocytes around the tumor, thus preventing liver injury. Furthermore, in view of the correlation between HMGB1 and adverse effects on tumor invasion and metastasis, it is proposed that NecroX-7 ameliorates the malignant potentials of hepatobiliary and pancreas cancers, which is a further focus of research. In conclusion, we demonstrated that the cell death pathway in massive IR injury is necrosis dominant and that the novel compound, NecroX-7, prevents hepatic IR injuryeinduced lethality, even at concentrations as low as 4.5 mg/kg, via reduction of HMGB1 release in a large animal setting. Thus, NecroX-7 may present an effective therapeutic option in a wide variety of human disorders, particularly in hepatobiliary diseases. The current findings provide a platform for advancing NecroX-7 to clinical trials for several human disease caused by IR injury. REFERENCES [1] Bilbao G, Contreras JL, Eckhoff DE, et al. Reduction of ischemia-reperfusion injury of the liver by in vivo adenovirusmediated gene transfer of the antiapoptotic Bcl-2 gene. Ann Surg 1999;230:185e93. [2] Degterev A, Yuan J. Expansion and evolution of cell death programmes. Nat Rev Mol Cell Biol 2008;9:378e90. [3] Clavien PA, Harvey PR, Strasberg SM. Preservation and reperfusion injuries in liver allografts. An overview and synthesis of current studies. Transplantation 1992;53:957e78. [4] Riordan S, Williams R. Bioartificial liver support: developments in hepatocyte culture and bioreactor design. Br Med Bull 1997;53:730e44. [5] Broelsch CE, Testa G, Alexandrou A, et al. Living related liver transplantation: medical and social aspects of a controversial therapy. Gut 2002;50:143e5. [6] Naruse K. Artificial liver support: future aspects. J Artif Organs 2005;8:71e6. [7] Naruse K, Tang W, Makuuch M. Artificial and bioartificial liver support: a review of perfusion treatment for hepatic failure patients. World J Gastroenterol 2007;13:1516e21. [8] Yu CB, Pan XP, Li LJ. Progress in bioreactors of bioartificial livers. Hepatobiliary Pancreat Dis Int 2009;8:134e40. [9] Delva E, Camus Y, Nordlinger B, et al. Vascular occlusions for liver resections. Operative management and tolerance to hepatic ischemia: 142 cases. Ann Surg 1989;209:211e8. [10] Elias D, Desruennes E, Lasser P. Prolonged intermittent clamping of the portal triad during hepatectomy. Br J Surg 1991;78: 42e4. [11] Hannoun L, Borie D, Delva E, et al. Liver resection with normothermic ischaemia exceeding 1 h. Br J Surg 1993;80:1161e5. [12] Belghiti J, Noun R, Malafosse R, et al. Continuous versus intermittent portal triad clamping for liver resection: a controlled study. Ann Surg 1999;229:369e75. [13] Man K, Fan ST, Ng IO, et al. Tolerance of the liver to intermittent pringle maneuver in hepatectomy for liver tumors. Arch Surg 1999;134:533e9.
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