Postischemic Liver Research Articles

SAT319 - The crucial role of PARP [poly (ADP-ribose polymerase] on the post-ischemic liver injury and inflammation

SAT319 - The crucial role of PARP [poly (ADP-ribose polymerase] on the post-ischemic liver injury and inflammation

Blockage of CX3CL1 Attenuates Platelet and Leukocyte Recruitment in Murine Hepatic I/R

Introduction: The chemokine fractalkine (CX3CL1) is critically involved in the pathophysiology of different inflammatory diseases and myocardial ischemia-reperfusion (I/R). This study aimed to analyze the role of CX3CL1 in the activation of platelets and leukocytes during hepatic I/R. Methods: Under inhalation anesthesia, C57BL6 mice were subjected to warm hepatic I/R (90 min/240 min). The animals were pretreated either with a function-blocking anti-mouse CX3CL1 antibody or IgG control administered systemically before ischemia. Sham-operated animals served as controls (n = 7 each group). The inflammatory response and sinusoidal perfusion failure were evaluated by intravital microscopy. Hepatic transaminases plasma levels and histopathological tissue damage were determined as markers of hepatocellular injury. Results: Sinusoidal perfusion failure, leukocyte recruitment to the liver, and transaminase activities were sharply increased upon I/R compared to sham-operated mice. Firm adhesion of platelets and concordantly leukocytes to endothelial cells is reduced significantly by a function-blocking anti-CX3CL1 antibody. We demonstrate that inhibition of CX3CL1 signaling attenuates leukocyte adhesion in the postischemic liver but does not significantly ameliorate overall perfusion failure and hepatocellular injury. Discussion/Conclusion: Our in vivo data demonstrate a mild attenuating effect of CX3CL1 blockade on platelet and leukocyte, but not CD4+ T cell accumulation and activation in hepatic I/R injury. We report a significant effect of blocking chemokine CX3CL1 on sinusoidal perfusion failure without considerably improving overall hepatocellular injury during early reperfusion.

Poly(ADP-ribose) Polymerase (PARP) is Critically Involved in Liver Ischemia/Reperfusion-injury

Poly(ADP-ribose) polymerase (PARP) is a DNA-repairing enzyme activated by extreme genomic stress, and therefore is potently activated in the remnant liver suffering from ischemia after surgical resection. However, the impact of PARP on post-ischemic liver injury has not been elucidated yet. We investigated the impact of PARP on murine hepatocyte/liver injury induced by hypoxia/ischemia, respectively. PJ34, a specific inhibitor of PARP, markedly protected against hypoxia/reoxygenation (H/R)-induced cell death, though z-VAD-fmk, a pan-caspase inhibitor similarly showed the protective effect. PJ34 did not affect H/R-induced caspase activity or caspase-mediated cell death. z-VAD-fmk also did not affect the production of PAR (i.e., PARP activity). Therefore, PARP- and caspase-mediated cell death occurred in a mechanism independent of each other in H/R. H/R immediately induced activation of PARP and cell death afterwards, both of which were suppressed by PJ34 or Trolox, an antioxidant. This suggests that H/R-induced cell death occurred redox-dependently through PARP activation. H/R and OS induced nuclear translocation of apoptosis inducing factor (AIF, a marker of parthanatos) and RIP1-RIP3 interaction (a marker of necroptosis), both of which were suppressed by PJ34. H/R induced PARP-mediated parthanatos and necroptosis redox-dependently. In mouse experiments, PJ34 significantly reduced serum levels of AST, ALT & LDH and areas of hepatic necrosis after liver ischemia/reperfusion, similar to z-VAD-fmk or Trolox. PARP, activated by ischemic damage and/or oxidative stress, may play a critical role in post-ischemic liver injury by inducing programmed necrosis (parthanatos and necroptosis). PARP inhibition may be one of the promising strategies against post-ischemic liver injury.

Macrophage extracellular traps aggravate iron overload-related liver ischaemia/reperfusion injury.

Macrophages regulate iron homeostasis in the liver and play important role in hepatic ischaemia/reperfusion (I/R) injury. This study investigates the role of macrophages in iron overload-related hepatocyte damage during liver I/R. Liver biopsies from patients undergoing partial hepatectomy with or without hepatic portal occlusion were recruited and markers of hepatocyte cell death and macrophage extracellular traps (METs) were detected. A murine hepatic I/R model was also established in high-iron diet-fed mice. Ferrostatin-1 and deferoxamine were administered to investigate the role of ferroptosis in hepatic I/R injury. The macrophage inhibitor liposome-encapsulated clodronate was used to investigate the interaction between macrophages and ferroptosis. AML12 hepatocytes and RAW264.7 macrophages were co-cultured in vitro. An inhibitor of macrophage extracellular traps was used to evaluate the role and mechanism of these traps and ferroptosis in hepatic I/R injury. Hepatocyte macrophage extracellular trap formation and ferroptosis were greater in patients who underwent hepatectomy with hepatic portal occlusion and in mice subjected to hepatic I/R. Macrophage extracellular traps increased when macrophages were subjected to hypoxia/reoxygenation and when they were co-cultured with hepatocytes. Ferroptosis increased and post-hypoxic hepatocyte survival decreased, which were reversed by inhibition of macrophage extracellular traps. Ferroptosis inhibition attenuated post-ischaemic liver damage. Moreover, iron overload induced hepatic ferroptosis and exacerbated post-ischaemic liver damage, which were reversed by the iron chelator. Macrophage extracellular traps are in volved in regulating ferroptosis highlighting the therapeutic potential of macrophage extracellular traps and ferroptosis inhibition in reducing liver I/R injury.

Hypoxia-inducible factor-1α-dependent induction of miR122 enhances hepatic ischemia tolerance.

Hepatic ischemia and reperfusion (IR) injury contributes to the morbidity and mortality associated with liver transplantation. microRNAs (miRNAs) constitute a family of noncoding RNAs that regulate gene expression at the posttranslational level through the repression of specific target genes. Here, we hypothesized that miRNAs could be targeted to enhance hepatic ischemia tolerance. A miRNA screen in a murine model of hepatic IR injury pointed us toward the liver-specific miRNA miR122. Subsequent studies in mice with hepatocyte-specific deletion of miR122 (miR122loxP/loxP Alb-Cre+ mice) during hepatic ischemia and reperfusion revealed exacerbated liver injury. Transcriptional studies implicated hypoxia-inducible factor-1α (HIF1α) in the induction of miR122 and identified the oxygen-sensing prolyl hydroxylase domain 1 (PHD1) as a miR122 target. Further studies indicated that HIF1α-dependent induction of miR122 participated in a feed-forward pathway for liver protection via the enhancement of hepatic HIF responses through PHD1 repression. Moreover, pharmacologic studies utilizing nanoparticle-mediated miR122 overexpression demonstrated attenuated liver injury. Finally, proof-of-principle studies in patients undergoing orthotopic liver transplantation showed elevated miR122 levels in conjunction with the repression of PHD1 in post-ischemic liver biopsies. Taken together, the present findings provide molecular insight into the functional role of miR122 in enhancing hepatic ischemia tolerance and suggest the potential utility of pharmacologic interventions targeting miR122 to dampen hepatic injury during liver transplantation.

Serp-2, a virus-derived apoptosis and inflammasome inhibitor, attenuates liver ischemia-reperfusion injury in mice

BackgroundIschemia-reperfusion injury (IRI) is an antigen-independent, innate immune response to arterial occlusion and ischemia with subsequent paradoxical exacerbation after reperfusion. IRI remains a critical problem after vessel occlusion and infarction or during harvest and surgery in transplants. After transplant, liver IRI (LIRI) contributes to increased acute and chronic rejection and graft loss. Tissue loss during LIRI has been attributed to local macrophage activation and invasion with excessive inflammation together with hepatocyte apoptosis and necrosis. Inflammatory and apoptotic signaling are key targets for reducing post-ischemic liver injury.Myxomavirus is a rabbit-specific leporipoxvirus that encodes a suite of immune suppressing proteins, often with extensive function in other mammalian species. Serp-2 is a cross-class serine protease inhibitor (serpin) which inhibits the inflammasome effector protease caspase-1 as well as the apoptotic proteases granzyme B and caspases 8 and 10. In prior work, Serp-2 reduced inflammatory cell invasion after angioplasty injury and after aortic transplantation in rodents. In this report, we explore the potential for therapeutic treatment with Serp-2 in a mouse model of LIRI.MethodsWildtype (C57BL/6 J) mice were subjected to warm, partial (70%) hepatic ischemia for 90 min followed by treatment with saline or Serp-2 or M-T7, 100 ng/g/day given by intraperitoneal injection on alternate days for 5 days. M-T7 is a Myxomavirus-derived inhibitor of chemokine-GAG interactions and was used in this study for comparative analysis of an unrelated viral protein with an alternative immunomodulating mechanism of action. Survival, serum ALT levels and histopathology were assessed 24 h and 10 days post-LIRI.ResultsSerp-2 treatment significantly improved survival to 85.7% percent versus saline-treated wildtype mice (p = 0.0135), while M-T7 treatment did not significantly improve survival (p = 0.2584). Liver viability was preserved by Serp-2 treatment with a significant reduction in serum ALT levels (p = 0.0343) and infarct scar thickness (p = 0.0016), but with no significant improvement with M-T7 treatment. Suzuki scoring by pathologists blinded with respect to treatment group indicated that Serp-2 significantly reduced hepatocyte necrosis (p = 0.0057) and improved overall pathology score (p = 0.0046) compared to saline. Immunohistochemistry revealed that Serp-2 treatment reduced macrophage infiltration into the infarcted liver tissue (p = 0.0197).ConclusionsTreatment with Serp-2, a virus-derived inflammasome and apoptotic pathway inhibitor, improves survival after liver ischemia-reperfusion injury in mouse models. Treatment with a cross-class immune modulator provides a promising new approach designed to reduce ischemia-reperfusion injury, improving survival and reducing chronic transplant damage.

РОЛЬ ГАЗОТРАНСМИТТЕРОВ (NO И H2S) В МЕХАНИЗМАХ ЗАЩИТЫ ОТ ПОСТИШЕМИЧЕСКИХ НАРУШЕНИЙ ПЕЧЕНИ

Postischemic liver injuries occur in clinical practice during liver transplantation, resection or trauma when afferent vessels should be occluded. The objective of the survey is to review the literature and our own data on the role of gasotransmitters (NO and H2S) in the mechanisms of liver protection during its ischemia-reperfusion syndrome.

Natural immunoglobulin M initiates an inflammatory response important for both hepatic ischemia reperfusion injury and regeneration in mice.

Complement plays a role in both hepatic ischemia reperfusion (IR) injury (IRI) and liver regeneration, but it is not clear how complement is activated in either process. We investigated the role of self-reactive immunoglobulin M (IgM) antibodies in activating complement after hepatic IR and liver resection. Natural IgM antibodies that recognize danger-associated molecular patterns (neoepitopes) activate complement following both hepatic IR and liver resection. Antibody-deficient Rag1-/- mice were protected from hepatic IRI, but had increased hepatic injury and an impaired regenerative response after 70% partial hepatectomy (PHx). We identified two IgM monoclonal antibodies (mAbs) that specifically reversed the effect of Rag1 deficiency in both models; B4 (recognizes Annexin IV) and C2 (recognizes subset of phospholipids). Focusing on the B4-specific response, we demonstrated sinusoidal colocalization of IgM and C3d in Rag1-/- mice that were reconstituted with B4 mAb, and furthermore that the Annexin IV neoepitope is specifically and similarly expressed after both hepatic IR and PHx in wild-type (WT) mice. A single-chain antibody construct (scFv) derived from B4 mAb blocked IgM binding and reduced injury post-IR in WT mice, although, interestingly, B4scFv did not alter regeneration post-PHx, indicating that anti-Annexin IV antibodies are sufficient, but not necessary, for the regenerative response in the context of an entire natural antibody repertoire. We also demonstrated expression of the B4 neoepitope in postischemic human liver samples obtained posttransplantation and a corollary depletion in IgM recognizing the B4 and C2 neoepitopes in patient sera following liver transplantation. Conclusion: These data indicate an important role for IgM in hepatic IRI and regeneration, with a similar cross-species injury-specific recognition system that has implications for the design of neoepitope targeted therapeutics. (Hepatology 2018;67:721-735).

In situ targeting of dendritic cells sets tolerogenic environment and ameliorates CD4+ T-cell response in the postischemic liver.

CD4+ T cells recruited to the liver play a key role in the pathogenesis of ischemia/reperfusion (I/R) injury. The mechanism of their activation during alloantigen-independent I/R is not completely understood. We hypothesized that liver-resident dendritic cells (DCs) interact with CD4+ T cells in the postischemic liver and that modulation of DCs or T-cell-DC interactions attenuates liver inflammation. In mice, warm hepatic I/R (90/120-240 min) was induced. Tolerogenic DCs were generated in situ by pretreatment of animals with the vitamin D analog paricalcitol. A mAb-CD44 was used for blockade of CD4+ T-cell-DC interactions. As shown by 2-photon in vivo microscopy as well as confocal microscopy, CD4+ T cells were closely colocalized with DCs in the postischemic liver. Pretreatment with paricalcitol attenuated I/R-induced maturation of DCs (flow cytometry), CD4+ T-cell recruitment into the liver (intravital microscopy), and hepatocellular/microvascular damage (intravital microscopy, alanine aminotransferase/aspartate aminotransferase, histology). However, interruption of T-cell-DC interaction increased proinflammatory DC maturation and even enhanced tissue damage. Simultaneous treatment with an anti-CD44mAb completely abolished the beneficial effect of paricalcitol on T-cell migration and tissue injury. Our study demonstrates for the first time that hepatic DCs interact with CD4+ T cells in the postischemic liver in vivo; modulation of DCs and/or generation of tolerogenic DCs attenuates intrahepatic CD4+ T-cell recruitment and reduces I/R injury; and interruption of CD44-dependent CD4+ T-cell-DC interactions enhances tissue injury by preventing the modulatory effect of hepatic DCs on T cells, especially type 1 T helper effector cells. Thus, hepatic DCs are strongly involved in the promotion of CD4+ T-cell-dependent postischemic liver inflammation.-Funken, D., Ishikawa-Ankerhold, H., Uhl, B., Lerchenberger, M., Rentsch, M., Mayr, D., Massberg, S., Werner, J., Khandoga, A. In situ targeting of dendritic cells sets tolerogenic environment and ameliorates CD4+ T-cell response in the postischemic liver.

Sirtuin 2 aggravates postischemic liver injury by deacetylating mitogen-activated protein kinase phosphatase-1.

Sirtuin 2 (Sirt2) is known to negatively regulate anoxia‐reoxygenation injury in myoblasts. Because protein levels of Sirt2 are increased in ischemia‐reperfusion (I/R)‐injured liver tissues, we examined whether Sirt2 is protective or detrimental against hepatic I/R injury. We overexpressed Sirt2 in the liver of C57BL/6 mice using a Sirt2 adenovirus. Wild‐type and Sirt2 knockout mice were subjected to a partial (70%) hepatic ischemia for 45 minutes, followed by various periods of reperfusion. In another set of experiments, wild‐type mice were pretreated intraperitoneally with AGK2, a Sirt2 inhibitor. Isolated hepatocytes and Kupffer cells from wild‐type and Sirt2 knockout mice were subjected to hypoxia‐reoxygenation injury to determine the in vitro effects of Sirt2. Mice subjected to I/R injury showed typical patterns of hepatocellular damage. Prior injection with Sirt2 adenovirus aggravated liver injury, as demonstrated by increases in serum aminotransferases, prothrombin time, proinflammatory cytokines, hepatocellular necrosis and apoptosis, and neutrophil infiltration relative to control virus‐injected mice. Pretreatment with AGK2 resulted in significant improvements in serum aminotransferase levels and histopathologic findings. Similarly, experiments with Sirt2 knockout mice also revealed reduced hepatocellular injury. The molecular mechanism of Sirt2's involvement in this aggravation of hepatic I/R injury includes the deacetylation and inhibition of mitogen‐activated protein kinase phosphatase‐1 and consequent activation of mitogen‐activated protein kinases. Conclusion: Sirt2 is an aggravating factor during hepatic I/R injury. (Hepatology 2017;65:225‐236).

Platelet aggregation but not activation and degranulation during the acute post-ischemic reperfusion phase in livers with no underlying disease

Platelets and P-selectin (CD62P) play an unequivocal role in the pathology of hepatic ischemia/reperfusion (I/R) injury. Inhibition or knock-out of P-selectin or immunodepletion of platelets results in amelioration of post-ischemic inflammation, reduced hepatocellular damage, and improved survival. However, P-selectin expression on platelets and endothelial cells, which concurs with platelet activation, has never been clearly demonstrated in I/R-subjected livers. To determine whether platelets become activated and degranulate in the acute phase of liver I/R and whether the platelets interact with neutrophils. Hepatic I/R was induced in male C57BL/6J mice (N = 12) using 37.5-min ischemia time. Platelets, endothelial cells, and neutrophils were fluorescently labeled by systemic administration of non-blocking antibodies. Cell kinetics were monitored by intravital spinning disk confocal microscopy during 90 min of reperfusion. Image analysis and quantification was performed with dedicated software. Platelets adhered to sinusoids more extensively in post-ischemic livers compared to livers not subjected to I/R and formed aggregates, which occurred directly after ischemia. Platelets and endothelial cells did not express P-selectin in post-ischemic livers. There was no interaction between platelets and neutrophils. Platelets aggregate but do not become activated and do not degranulate in post-ischemic livers. There is no platelet-neutrophil interplay during the early reperfusion phase in a moderate model of hepatic I/R injury. The mechanisms underlying the biological effects of platelets and P-selectin in this setting warrant further investigation. I/R in surgical liver patients may compromise outcome due to post-ischemic oxidative stress and sterile inflammation. Both processes are mediated in part by platelets. Understanding platelet function during I/R is key to developing effective interventions for I/R injury and improving clinical outcomes.

PP14 - Aggravation of post-ischemic liver injury by overexpression of insulin-like growth factor binding protein 3

Insulin-like growth factor-1 (IGF-1) is known to inhibit reperfusion-induced apoptosis, thus preventing hepatic ischemia/reperfusion (I/R) injury. IGF-binding protein-3 (IGFBP-3) is the major circulating carrier protein for IGF-1 and induces apoptosis. In this study, we determined if IGFBP-3 was important in the hepatic response to I/R. To deliver IGFBP-3, we used an adenovirus containing IGFBP-3 cDNA (AdIGFBP-3) or an IGFBP-3 mutant devoid of IGF binding affinity but retaining IGFBP-3 receptor binding ability (AdIGFBP-3GGG). Mice subjected to I/R injury showed typical patterns of hepatocellular damage. Protein levels of IGFBP-3 were increased after reperfusion and showed a positive correlation with the extent of liver injury. Prior injection with AdIGFBP-3 aggravated liver injury: serum aminotransferases, prothrombin time, proinflammatory cytokines, hepatocellular necrosis and apoptosis, and neutrophil infiltration were markedly increased compared to control mice. A decrease in antioxidant potential and an upregulation of NADPH oxidase might have caused these aggravating effects of IGFBP-3. Experiments using HepG2 cells and N-acetylcysteine-pretreated mice showed a discernible effect of IGFBP-3 on reactive oxygen species generation. Lastly, AdIGFBP-3 abolished the beneficial effects of ischemic preconditioning and hypothermia. Mice treated with AdIGFBP-3GGG exhibited effects similar to those of AdIGFBP3, suggesting a ligand-independent effect of IGFBP-3. Our results suggest IGFBP-3 as an aggravating factor during hepatic I/R injury.

RIP1-Dependent Programmed Necrosis is Negatively Regulated by Caspases During Hepatic Ischemia-Reperfusion.

Programmed necrosis (necroptosis), a newly discovered form of cell death, is mediated by receptor-interacting protein 1 (RIP1) and plays a pivotal role after myocardial, renal, and cerebral ischemia-reperfusion (I/R). The relevance of necroptosis in the postischemic liver remains, however, unclear. The aim of this study was to analyze the role of programed necrosis during hepatic I/R. C57BL6 mice were subjected to warm hepatic I/R (90 min/240 min). The animals were pretreated with either the RIP1 inhibitor necrostatin-1 (Nec-1, 3.5 μg kg) or vehicle (Nec-1inactive, 3.5 μg kg) administered systemically before ischemia. Sham-operated animals served as controls (n = 6 each group). The inflammatory response was evaluated by intravital microscopy. The hepatic transaminases alanine aminotransferase/aspartate aminotransferase in plasma as well as the activity of caspase-3 in tissue were determined as markers of hepatocellular injury. Leukocyte recruitment to the liver, sinusoidal perfusion failure, as well as the transaminase activities were strongly increased on I/R as compared with the sham-operated mice. Inhibition of the RIP1-dependent pathway with Nec-1, however, did not attenuate I/R-induced leukocyte migration, perfusion failure, and hepatocellular injury. Western blot analysis showed a baseline RIP1 expression in livers from sham-operated mice, whereas RIP1 expression was not detectable in both Nec-1-treated and vehicle-treated I/R group. Caspase-3 activity was significantly elevated after I/R in both postischemic groups. Our in vivo data show that RIP1-mediated necroptosis is not present in the postischemic liver and that I/R-induced caspase activation is associated with loss of RIP1 expression. Because caspases are able to cleave RIP1, we hypothesize that I/R-triggered caspase activation negatively regulates necroptosis and, thereby, determines apoptosis as a preferred route of cell death after hepatic I/R.

Aggravation of post-ischemic liver injury by overexpression of insulin-like growth factor binding protein 3.

Insulin-like growth factor-1 (IGF-1) is known to inhibit reperfusion-induced apoptosis. IGF-binding protein-3 (IGFBP-3) is the major circulating carrier protein for IGF-1 and induces apoptosis. In this study, we determined if IGFBP-3 was important in the hepatic response to I/R. To deliver IGFBP-3, we used an adenovirus containing IGFBP-3 cDNA (AdIGFBP-3) or an IGFBP-3 mutant devoid of IGF binding affinity but retaining IGFBP-3 receptor binding ability (AdIGFBP-3GGG). Mice subjected to I/R injury showed typical patterns of hepatocellular damage. Protein levels of IGFBP-3 were increased after reperfusion and showed a positive correlation with the extent of liver injury. Prior injection with AdIGFBP-3 aggravated liver injury: serum aminotransferases, prothrombin time, proinflammatory cytokines, hepatocellular necrosis and apoptosis, and neutrophil infiltration were markedly increased compared to control mice. A decrease in antioxidant potential and an upregulation of NADPH oxidase might have caused these aggravating effects of IGFBP-3. Experiments using HepG2 cells and N-acetylcysteine-pretreated mice showed a discernible effect of IGFBP-3 on reactive oxygen species generation. Lastly, AdIGFBP-3 abolished the beneficial effects of ischemic preconditioning and hypothermia. Mice treated with AdIGFBP-3GGG exhibited effects similar to those of AdIGFBP-3, suggesting a ligand-independent effect of IGFBP-3. Our results suggest IGFBP-3 as an aggravating factor during hepatic I/R injury.

Modulating CD4+ T cell migration in the postischemic liver: hepatic stellate cells as new therapeutic target?

CD4+ T cells play a critical role during hepatic ischemia-reperfusion (I/R) injury although the mechanisms of their migration in the postischemic liver remain unclear. We answered the questions of whether hepatic stellate cells (HSCs) interact with CD4+ T cells during I/R of the liver and whether modulation of HSC activity affects T cell-dependent I/R injury. In mice, migration of CD4+ T cells was analyzed in vivo using conventional intravital microscopy and two-photon microscopy. CD4+ T cell-HSC interactions were visualized after infusion of fluorescence-labeled CD4+ T cells into Cx3CR1 mice (mice exhibiting GFP-labeled HSCs) after I/R. Because the activation of HSC is controlled by endocannabinoid receptors, CB-1 and CB-2, the mice received treatment before I/R with the CB-2 agonist JWH-133 to reach HSC depletion or the CB-1 agonist arachidonylcyclopropylamide to activate HSCs. Sinusoidal perfusion and liver transaminases were used as markers of I/R injury. Hepatic I/R induced CD4+ T cell recruitment in sinusoids. More than 25% of adherent CD4+ T cells were colocalized with HSCs during reperfusion, suggesting a direct cell-cell interaction. The HSC deactivation with JWH-133 significantly attenuated the CD4+ T cell recruitment in the postischemic liver and reduced I/R injury as compared to the vehicle-treated group. The HSC hyperactivation by CB-1, however, did not affect T-cell migration and even increased perfusion failure. Our in vivo data suggest that CD4+ T cells interact with HSCs on their migration into the hepatic parenchyma, and a depletion or deactivation of HSCs protects the liver from T cell-dependent I/R injury.

Preconditioning by cilostazol protects against cold hepatic ischemia-reperfusion injury.

In liver transplantation, prolonged cold storage and post-transplant reperfusion are associated with endothelial inflammation, organ dysfunction, and graft failure. We herein studied whether cilostazol, a phosphodiesterase-3-inhibitor, attenuates post-ischemic liver injury after prolonged cold storage. Sprague-Dawley rats were assigned to 5 groups (n=6 each): sham animals (cold storage time (CST): 1 h), vehicle-treated (NaCl 0.9%) controls (CST: 24 h), and animals receiving 0.1, 1.0, or 10.0 mg/kg body weight (BW) cilostazol pre-treatment (CST: 24 h). After organ explantation, all livers were stored at 4°C in HTK solution, followed by 60 min of reperfusion with 37°C Krebs Henseleit buffer in a non-recirculating ex situ system. Bile flow was measured to evaluate liver function. To analyze inflammation and morphology, liver tissue samples were taken and histology, immunohistochemistry, and Western blotting were conducted. In vehicle-treated controls, prolonged cold storage and warm reperfusion induced inflammation, organ dysfunction, and cell death. This was indicated by an increase of hepatocellular vacuolization, endothelial ICAM-1 expression, and apoptotic cell death compared to sham animals. Cilostazol pre-treatment protected against cold hepatic ischemia-reperfusion injury by preventing hepatocellular disintegration, ICAM-1-associated endothelial inflammation, and apoptotic death. Inhibition of PDE3 reduces endothelial cell activation and hepatocellular injury in cold ischemia/reperfusion of the liver.

Hepatocellular Heme Oxygenase-1

Hepatic ischemia-reperfusion (IR) results in progressive injury; initiated by oxidative stress during ischemia and compounded by cytokine-mediated inflammation during reperfusion. Recovery requires strict regulation of these events. Recombinant human erythropoietin (rhEPO) is thought to mitigate hepatocellular IR injury by altering the nonparenchymal liver microenvironment. This study sought to identify additional mechanisms whereby rhEPO is protective after liver IR injury. Mice were treated with rhEPO (4 units/g s.c.) at the onset of partial liver ischemia and assessed for transaminase and histologic injury at intervals after reperfusion. Induction of cytokines, activation of signal transducers and activators of transcription (STATs), suppressors of cytokine signaling (Socs1, Socs3, Cis), caspase-3 activation, and heme oxygenase-1 (HO-1) expression were assessed in postischemic liver. Effects of rhEPO stimulation were further characterized in whole-liver lysates from mice undergoing rhEPO injection alone and in cultured AML-12 hepatocytes. Recombinant human erythropoietin treatment at the onset of severe (90 min) hepatic IR confirmed commensurate biochemical and histological protection without affecting tissue cytokine levels. Although Socs3 and STAT5 activation were induced in normal liver after in vivo rhEPO injection, this treatment did not augment expression beyond that seen with IR alone, and neither was induced in cultured hepatocytes treated with rhEPO. Recombinant human erythropoietin inhibited caspase-3 activation in nonparenchymal cells, whereas hepatocellular HO-1 was rapidly induced both in vivo and in vitro with rhEPO treatment. These data suggest HO-1 as a potent mechanism of rhEPO-mediated protection after liver IR, which involves both direct hepatocellular and nonparenchymal mechanisms.

Biomarkers distinguish apoptotic and necrotic cell death during hepatic ischemia/reperfusion injury in mice.

Hepatic ischemia/reperfusion (IRP) injury is a significant clinical problem during tumor-resection surgery (Pringle maneuver) and liver transplantation. However, the relative contribution of necrotic and apoptotic cell death to the overall liver injury is still controversial. To address this important issue with a standard murine model of hepatic IRP injury, plasma biomarkers of necrotic cell death such as micro-RNA 122, full-length cytokeratin 18 (FK18), and high-mobility group box 1 (HMGB1) protein and plasma biomarkers of apoptosis such as plasma caspase-3 activity and caspase-cleaved fragment of cytokeratin 18 (CK18) coupled with markers of inflammation (hyperacetylated HMGB1) were compared by histological features in hematoxylin and eosin-stained and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-stained liver sections. After 45 minutes of hepatic ischemia and 1 to 24 hours of reperfusion, all necrosis markers increased dramatically in plasma by 40- to >10,000-fold over the baseline with a time course similar to that of alanine aminotransferase. These data correlated well with histological characteristics of necrosis. Within the area of necrosis, most cells were TUNEL positive; initially (≤3 hours of reperfusion), the staining was restricted to nuclei, but it later spread to the cytosol, and this is characteristic of karyorrhexis during necrotic cell death. In contrast, the lack of morphological evidence of apoptotic cell death and relevant caspase-3 activity in the postischemic liver correlated well with the absence of caspase-3 activity and CK18 (except for a minor increase at 3 hours of reperfusion) in plasma. A quantitative comparison of FK18 (necrosis) and CK18 (apoptosis) release indicated dominant cell death by necrosis during IRP and only a temporary and very minor degree of apoptosis. These data suggest that the focus of future research should be the elucidation of necrotic signaling mechanisms to identify relevant targets, which may be used to attenuate hepatic IRP injury.

Augmenter of liver regeneration attenuates inflammatory response in the postischemic mouse liver in vivo

BackgroundAugmenter of Liver Regeneration (ALR), a protein synthesized in the liver is suggested to be protective against oxidative stress–induced cell death. Hepatic ischemia–reperfusion (I/R) injury is triggered by reactive oxygen species. Here, we tested the hypothesis that ALR attenuates hepatic I/R injury in vivo. MethodsC57BL6 mice were subjected to warm hepatic ischemia for 90 min. Either recombinant ALR (100 μg/kg) or vehicle were administered to mice prior ischemia. During reperfusion, neutrophil and CD4+ T cell migration and sinusoidal perfusion were analyzed using intravital microscopy. Alanine aminotransferase–aspartate aminotransferase (plasma) and caspase-3 (tissue) activities were determined as markers of hepatocellular necrotic and apoptotic injury. ResultsHepatic I/R led to dramatic enhancement of neutrophil and CD4+ T cell recruitment in hepatic microvessels, sinusoidal perfusion failure, and strong elevation of aspartate aminotransferase–alanine aminotransferase and caspase-3 activities. During early reperfusion (60 min), the pretreatment with ALR improved postischemic perfusion failure (P < 0.05) and attenuated liver enzyme activities. Recruitment of CD4+ T cells, but not of neutrophils was attenuated. After 240 min of reperfusion, the protective effect of ALR was stronger, since the liver enzyme activity, perfusion failure, and leukocyte influx were significantly attenuated. As shown by the measurement of caspase-3 activity, postischemic apoptosis was reduced in the ALR-treated group. ConclusionsOur in vivo data show that ALR has a therapeutic potential against postischemic liver injury. As a mechanism, we suggest a direct protective effect of ALR on apoptotic and necrotic death of hepatocytes and an attenuation of inflammatory cell influx into the postischemic tissue.

Targeting Platelet Migration in the Postischemic Liver by Blocking Protease-Activated Receptor 4

Platelets play a critical role during hepatic ischemia/reperfusion (I/R). Antiplatelet strategies during liver transplantation are, however, limited because of bleeding complications. Thrombin is activated during reperfusion and regulates platelet and endothelial cell function via protease-activated receptor 4 (PAR-4). Interventions at the level of PAR-4, the main platelet receptor for thrombin, are assumed to attenuate the proinflammatory effects of thrombin without affecting blood coagulation. The aim of our study was to analyze the impact of PAR-4 blockade on platelet recruitment and microvascular injury during hepatic I/R. C57BL/6 mice undergoing hepatic I/R (90 min/60 min and 240 min) were treated either with a selective PAR-4 antagonist TcY-NH2 or vehicle. Sham-operated animals served as controls. Recruitment of freshly isolated and fluorescence-labeled platelets and CD4 T cells was analyzed using intravital video fluorescence microscopy. Parameters of tissue injury, regeneration, and blood coagulation were assessed in tissue/blood samples. Results show that treatment with TcY-NH2 attenuated I/R-induced platelet and CD4 T-cell recruitment, improved sinusoidal perfusion failure, and reduced apoptotic and necrotic injury. The protective effect of PAR-4 blockade did not suppress hemostasis or liver regeneration. Our in vivo data suggest PAR-4 as a potential target for future therapeutic strategies against platelet-mediated liver injury on transplantation.