Ischemia-reperfusion Injury In Liver Transplantation Research Articles

Differential Effects of HDAC6 Inhibition Versus Knockout During Hepatic Ischemia-Reperfusion Injury Highlight Importance of HDAC6 C-terminal Zinc-finger Ubiquitin-binding Domain.

Ischemia-reperfusion injury (IRI) causes significant morbidity in liver transplantation among other medical conditions. IRI following liver transplantation contributes to poor outcomes and early graft loss. Histone/protein deacetylases (HDACs) regulate diverse cellular processes, play a role in mediating tissue responses to IRI, and may represent a novel therapeutic target in preventing IRI in liver transplantation. Using a previously described standardized model of murine liver warm IRI, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were assessed at 24 and 48 h after reperfusion to determine the effect of different HDAC inhibitors. Broad HDAC inhibition with trichostatin-A (TSA) was protective against hepatocellular damage ( P < 0.01 for AST and P < 0.05 for ALT). Although HDAC class I inhibition with MS-275 provided statistically insignificant benefit, tubastatin-A (TubA), an HDAC6 inhibitor with additional activity against HDAC10, provided significant protection against liver IRI ( P < 0.01 for AST and P < 0.001 for ALT). Surprisingly genetic deletion of HDAC6 or -10 did not replicate the protective effects of HDAC6 inhibition with TubA, whereas treatment with an HDAC6 BUZ-domain inhibitor, LakZnFD, eliminated the protective effect of TubA treatment in liver ischemia ( P < 0.01 for AST and P < 0.01 for ALT). Our findings suggest TubA, a class IIb HDAC inhibitor, can mitigate hepatic IRI in a manner distinct from previously described class I HDAC inhibition and requires the HDAC6 BUZ-domain activity. Our data corroborate previous findings that HDAC targets for therapeutic intervention of IRI may be tissue-specific, and identify HDAC6 inhibition as a possible target in the treatment of liver IRI.

Wogonin Alleviates DCD Liver Ischemia/Reperfusion Injury by Regulating ALOX15/iNOS-mediated Ferroptosis.

Donation after circulatory death livers are more susceptible to ischemia/reperfusion injury (IRI) because of a longer period of warm ischemia. Growing evidence now suggests that ferroptosis plays a key regulatory role in the development of IRI, so targeting ferroptosis may be an effective strategy to alleviate IRI in liver transplantation (LT). Using donation after circulatory death LT models in rats and oxygen-glucose deprivation/reoxygenation (OGD/R) models in BRL-3A cells, we tested the effect of the Chinese medicine monomer wogonin on liver IRI and explored the specific mechanism. Wogonin attenuated liver IRI and increased the survival rate of rats by inhibiting lipid peroxidation and ferroptosis. Mechanistically, arachidonic acid 15-lipoxygenase-1 (ALOX15) and inducible nitric oxide synthase (iNOS) were identified as potential targets of baicalein through bioinformatics analysis combined with in vivo and in vitro experiments. This result was further confirmed by molecular docking and cellular thermal shift assays. Finally, we silenced ALOX15 and iNOS in the OGD/R cell model and found that silencing ALOX15 and iNOS could reproduce the regulatory effect of wogonin and abrogate the regulatory effect of wogonin. In brief, this study emphasizes that wogonin exerts a protective effect in liver IRI through the regulation of ALOX15- and iNOS-mediated ferroptosis. ALOX15 and iNOS are potential targets for intervention in IRI induced by LT, and wogonin is a drug candidate for LT patients.

Human umbilical cord mesenchymal stem cells alleviate fatty liver ischemia-reperfusion injury by activating autophagy through upregulation of IFNγ.

Liver ischemia-reperfusion injury (IRI) is an important factor affecting the prognosis of liver transplantation, and extended criteria donors (e.g., steatosis donor livers) are considered to be more sensitive to ischemia-reperfusion injury in liver transplantation. Currently, the application of human umbilical cord mesenchymal stem cells (hMSCs) has great promise in the treatment of various injuries in the liver. This study aimed to investigate the therapeutic role and mechanism of hMSCs in fatty liver IRI. After more than 8 weeks of high-fat chow feeding, we constructed a fatty liver mouse model and established ischemic injury of about 70% of the liver. Six hours after IRI, liver injury was significantly alleviated in hMSCs-treated mice, and the expression levels of liver enzyme, inflammatory factor TNF-α, and apoptotic proteins were significantly lower than those of the control group, which were also significant in pathological sections. Transcriptomics analysis showed that IFNγ was significantly upregulated in the hMSCs group. Mechanistically, IFNγ, which activates the MAPK pathway, is a potent agonist that promotes the occurrence of autophagy in hepatocytes to exert a protective function, which was confirmed by in vitro experiments. In summary, hMSCs treatment could slow down IRI in fatty liver by activating autophagy through upregulation of IFNγ, and this effect was partly direct.

Bone marrow-derived mesenchymal stem cells reactive oxygen species-responsively secreting hepatocyte growth factor for effective treatment of ischemia-reperfusion injury in liver transplantation

Bone marrow-derived mesenchymal stem cells reactive oxygen species-responsively secreting hepatocyte growth factor for effective treatment of ischemia-reperfusion injury in liver transplantation

N-acetylcysteine and Reduction of Ischemia-reperfusion Injury in Liver Transplantation.

In the current issue of Transplantation, Jia et al perform a systemic review and meta-analysis looking at the utilization of N-acetylcysteine (NAC) in the donor and recipient in liver transplantation.1,2 It has been suggested that NAC may have a role in mitigating ischemia-reperfusion injury that occurs during the various stages of organ donation. The pathophysiology of ischemia-reperfusion injury is mainly due to the oxidative stress and cytotoxicity arising from the imbalance between oxidants and antioxidants. NAC is a rich source of sulfhydryl groups, which are important for replenishing glutathione (GSH) stores. GSH acts as a free radical scavenger to decrease damage caused by the toxic free radicals. GSH is the main endogenous antioxidant, and the liver is the main organ producing GSH under normal conditions.2 The present meta-analysis demonstrates a lower incidence of primary nonfunction, a reduction in posttransplant peak aspartate transaminase and alanine transaminase, and reduced postoperative complications in the NAC treatment groups compared with the control groups. As a long-time proponent of the use of NAC in the transplant setting, the overall results of this meta-analysis are encouraging. At Mayo Clinic Florida, we routinely use a NAC infusion for donation after circulatory death (DCD) liver transplant recipients. Our protocol involves a bolus of 150 mg/kg given over 1 h before reperfusion followed by an infusion of 150 mg/kg given for 72 h. Our current rate of ischemic cholangiopathy is <4%; however, whether NAC provides any direct benefit in the reduction of ischemic cholangiopathy remains unproven.3 More recently, we have also started using NAC in the perfusate when performing abdominal normothermic regional perfusion in DCD donors ([6 g/30 mL] × 3 vials). Negative effects of NAC administration are minimal. Although the meta-analysis demonstrated a slightly increased need for packed red blood cells and cryoprecipitate in NAC treatment groups, the magnitude of difference was <1 unit. This may imply that although a statistically significant difference was seen, such a small difference may not be clinically significant. There are some limitations to the present study that should be mentioned. One of the challenges with combining the data from the different studies in the present analysis is that some of the studies gave NAC to the donors at the time of donor procurement, some gave NAC to the recipients at the time of transplant, and others gave NAC to both the donor and recipient. Dosing and timing of dosing was also very heterogeneous between the different studies. Given this heterogeneity, developing a comprehensive protocol utilizing NAC is problematic. In the subanalysis, the authors did suggest that NAC administration to the recipient appeared to produce better clinical effects than administration to the donor. Another unanswered question based on the present analysis is how the benefits of NAC may vary by donor type? Some of the studies looked at living donors while others looked at donation after brain death donors and others looked at DCD donors. Could there be a more significant effect in marginal liver grafts with a suspected higher degree of ischemia-reperfusion injury or should NAC be utilized in all cases? Finally postoperative complications were treated as a binary variable (yes/no) for the analysis. Given the vastly different implications of complications based on their severity, using a classification system such as Clavien-Dindo would be more meaningful when comparing study groups. Despite the limitations of the present meta-analysis, the results are encouraging and suggest that NAC does have some potentially beneficial effects in reducing ischemia-reperfusion injury when used in the setting of liver transplantation. The adequate safety of NAC, in addition to its low costs, makes it an appealing intervention. Further studies are needed before evidence is strong enough to recommend a specific protocol describing both optimal timing and dosage for administration. Moreover, the donor type that may achieve the greatest benefit remains unknown.

Donor graft METTL3 gene transfer ameliorates rat liver transplantation ischemia-reperfusion injury by enhancing HO-1 expression in an m6A-dependent manner

Ischemia-reperfusion injury (IRI) is one of the most common complications in liver transplantation. METTL3 regulates inflammation and cellular stress response by modulating RNA m6A modification level. Here, the study aimed to investigate the role and mechanism of METTL3 in IRI after rat orthotopic liver transplantation. The total RNA m6A modification and METTL3 expression level was consistently down-regulated after 6 h or 24 h reperfusion in OLT, which is negatively associated with the hepatic cell apoptosis. Functionally, METTL3 pretreatment in donor significantly inhibited liver grafts apoptosis, improved liver function and depressed the proinflammatory cytokine/chemokine expression. Mechanistically, METTL3 inhibited apoptosis of grafts via upregulating HO-1. Moreover, m6A dot blot and MeRIP-qPCR assay revealed that METTL3 promoted HO-1 expression in an m6A-dependent manner. In vitro, METTL3 alleviated hepatocytes apoptosis by upregulating HO-1 under hypoxia/reoxygenation condition. Taken together, these findings demonstrate that METTL3 ameliorates rat OLT-stressed IRI by inducing HO-1 in an m6A-dependent manner, highlighting a potential target for IRI in liver transplantation.

Retrograde reperfusion of renal grafts to reduce ischemic-reperfusion injury.

During transplantation, a kidney graft undergoes a cascade of pathological changes, referred to as ischemia-reperfusion injury (IRI), as it is incorporated into the bloodstream. Various studies have reported that retrograde reperfusion (RRP) leads to improved myocardial recovery and could reduce IRI in liver transplantation. This study investigated the effect of RRP in renal transplantation with a focus on reduction of kidney IRI. Between December 2019 and July 2022, 15 consecutive kidney transplants were performed with retrograde venous reperfusion. To conduct a comparative study and to recruit a control group, 15 kidney transplants that had been performed in the same center by the same two surgeons were retrospectively analyzed. Differences between the two groups were considered statistically significant at P<0.05. The baseline characteristics of the two groups were statistically comparable (P>0.05). The surgical technique for kidney transplantation was the same in both groups. On the first postoperative day, polyuria was less pronounced in the RRP group (P<0.01). Serum creatinine and urea levels and estimated glomerular filtration rates on postoperative days 1, 4, 7, and 30 were lower in the RRP group (P<0.05). Retrograde venous reperfusion of a kidney transplant, preceding antegrade arterial reperfusion, reduced the effects of renal parenchyma IRI. To validate the results of this study, it is necessary to conduct further studies on a larger cohort of patients with a longer follow-up period.

α-Connexin Carboxyl Terminal Peptide 1 Attenuates Ischemia-Reperfusion Injury in Liver Transplantation With Extended Cold Preservation by Stabilizing Cell Junctions in Mice

• ⚫Peptide α-connexin carboxyl terminal peptide (ACT1) protects graft livers during cold storage by steadying cell junctions. • ⚫ACT1 reduces hepatic ischemia-reperfusion injury following transplantation. • ⚫ACT1 inhibits acute and chronic inflammation after transplantation. • ⚫The effect of ACT1 on cell junctions is long term. Ischemia-reperfusion injury is an inevitable process that the liver must go through after liver transplantation. Because of the worldwide shortage of grafts, reliable intervention strategies should be explored to protect grafts. α-Connexin carboxyl terminal peptide (ACT1), which is a synthetic peptide containing 25 amino acids, has the effect of stabilizing cell junctions. The purpose of this study was to investigate the role of ACT1 on ischemia-reperfusion injury after liver transplantation in mice. Experimental results showed that injury to livers that underwent extended cold preservation was reduced by ACT1. After 6 hours of reperfusion, ACT1-treated livers showed less necrosis and acute inflammation and the cell junctions were strengthened. Moreover, after 28 days of reperfusion, the application of ACT1 distinctly alleviated hepatic damage and later cholangiopathy as well as conspicuously consolidated the cell junctions. This study indicated that ACT1 produced affirmative effects in suppressing hepatic ischemia-reperfusion injury in liver transplantation with extended cold preservation in mice by protecting cell junctions and may be a potential strategy to extend the cold preservation time of grafts for liver transplantation.

Iron overload as a risk factor for hepatic ischemia-reperfusion injury in liver transplantation: Potential role of ferroptosis.

Hepatic ischemia-reperfusion (I/R) injury is a major problem in liver transplantation (LT). Although hepatocyte cell death is the initial event in hepatic I/R injury, the underlying mechanism remains unclear. In the present study, we retrospectively analyzed the clinical data of 202 pediatric living donor LT and found that a high serum ferritin level, a marker of iron overload, of the donor is an independent risk factor for liver damage after LT. Since ferroptosis has been recently discovered as an iron-dependent cell death that is triggered by a loss of cellular redox homeostasis, we investigated the role of ferroptosis in a murine model of hepatic I/R injury, and found that liver damage, lipid peroxidation, and upregulation of the ferroptosis marker Ptgs2 were induced by I/R, and all of these manifestations were markedly prevented by the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) or α-tocopherol. Fer-1 also inhibited hepatic I/R-induced inflammatory responses. Furthermore, hepatic I/R injury was attenuated by iron chelation by deferoxamine and exacerbated by iron overload with a high iron diet. These findings demonstrate that iron overload is a novel risk factor for hepatic I/R injury in LT, and ferroptosis contributes to the pathogenesis of hepatic I/R injury.

Comprehensive and combined omics analysis reveals factors of ischemia-reperfusion injury in liver transplantation.

To explore molecular mechanisms underlying liver ischemia-reperfusion injury (IRI). Four Gene Expression Omnibus datasets comprising liver transplantation data were collected for a comprehensive analysis. A proteomic analysis was performed and used for correlations analysis with transcriptomic. Ten differentially expressed genes were co-upregulated in four Gene Expression Omnibus datasets, including ATF3, CCL4, DNAJB1, DUSP5, JUND, KLF6, NFKBIA, PLAUR, PPP1R15A and TNFAIP3. The combined analysis demonstrated ten coregulated genes/proteins, including HBB, HBG2, CA1, SLC4A1, PLIN2, JUNB, HBA1, MMP9, SLC2A1 and PADI4. The coregulated differentially expressed genes and coregulated genes/proteins formed a tight interaction network and could serve as the core factors underlying IRI. Comprehensive and combined omics analyses revealed key factors underlying liver IRI, and thus having potential clinical significance.

Normothermic Machine Perfusion of Deceased Donor Liver Grafts Is Associated With Improved Postreperfusion Hemodynamics.

BackgroundGraft reperfusion poses a critical challenge during liver transplantation and can be associated with hemodynamic instability/postreperfusion syndrome. This is sequel to ischemia-reperfusion injury and normothermic machine preservation (NMP) may affect hemodynamic changes. Herein, we characterize postreperfusion hemodynamics in liver grafts after NMP and traditional cold preservation.Materials and methodsIntraoperative records of patients receiving grafts after NMP (n = 6; NMP group) and cold storage (CS) (n = 12; CS group) were compared. The mean arterial pressure (MAP) was defined as the average pressure in the radial artery during 1 cardiac cycle by invasive monitoring. Postreperfusion syndrome was defined as MAP drop greater than 30% of baseline, lasting for 1 minute or longer within the first 5 minutes from graft reperfusion.ResultsDonor, recipient, demographics, and surgical parameters were evenly matched. Normothermic machine preservation grafts were perfused for 525 minutes (395-605 minutes) after initial cold ischemic time of 91 minutes (73-117 minutes), whereas in CS group cold ischemic time was 456 minutes (347-685 minutes) (P = 0.001). None developed postreperfusion syndrome in the NMP group against n = 2 (16.7%) in CS group (P = 0.529). Normothermic machine preservation group had better intraoperative MAP at 90 minutes postreperfusion (P = 0.029), achieved with a significantly less vasopressor requirement (P = <0.05) and less transfusion of blood products (P = 0.030) compared with CS group.ConclusionsNormothermic machine perfusion is associated with a stable intraoperative hemodynamic profile postreperfusion, requiring significantly less vasopressor infusions and blood product transfusion after graft reperfusion and may have benefit to alleviate ischemia-reperfusion injury in liver transplantation.

Isoflurane does not protect from brain death-associated aggravation of cold hepatic ischemia-reperfusion injury.

Previous studies have shown that brain death aggravates cold ischemia-reperfusion injury in liver transplantation. Isoflurane, a volatile anesthetic, has been indicated to reduce warm hepatic ischemia-reperfusion injury. Herein, we studied in Sprague-Dawley rats whether isoflurane is capable of ameliorating brain death-associated aggravation of cold hepatic ischemia-reperfusion injury. Brain-dead animals were treated for 30 min with isoflurane (MAC 1.5%; n=8). Animals without isoflurane treatment served as controls (n=8). Another 13 animals without induction of brain death served as sham controls. After a 4-h period portal venous blood perfusion, hepatic microcirculation and bile flow were determined. Livers were recovered and stored for 24 h in 4°C cold HTK solution, followed by reperfusion with 37°C Krebs-Henseleit-buffer for 60 min. Liver enzymes in the effluent and bile flow were analyzed. Hepatocellular morphology was determined by histology and immunohistochemistry. Brain death reduced portal venous blood perfusion and bile flow, induced heme oxygenase-1 (HO-1), and resulted in hepatocellular damage. Isoflurane treatment did not prevent the reduction of portal venous blood perfusion or bile flow or the induction of HO-1. Accordingly, isoflurane was not capable of reducing the hepatocellular injury. Isoflurane does not protect from brain death-associated aggravation of cold hepatic ischemia-reperfusion injury.

Impact of brain death on ischemia/reperfusion injury in liver transplantation

In liver transplantation, the ischemia/reperfusion injury (IRI) is influenced by factors related to graft quality, organ procurement and the transplant procedure itself. However, in brain-dead donors, the process of death itself also thoroughly affects organ damage through breakdown of the autonomous nervous system and subsequent massive cytokine release. This review highlights the actual knowledge on these proinflammatory effects of brain death on IRI in liver transplantation. Brain death affects IRI either through hemodynamical or molecular effects with proinflammatory activation. Immunological effects are mainly mediated through Kupffer cell activation, leading to TNF-α and TLR4 amplification. Proinflammatory cytokines such as interleukin (IL)-6, IL-10, TNF-β and MIP-1α are released, together with activation of the innate immune system via natural killer cells and natural killer T cells, which promote organ damage and activation of fibrosis. Preprocurement treatment regimens attempt to hamper inflammatory response by the application of methylprednisolone or thymoglobulin to the donor. Selective P-selectin antagonism resulted in improved function in marginal liver grafts. Inhaled nitric oxide was found to reduce apoptosis in liver grafts. Other medications like the immunosuppressant tacrolimus produced conflicting results regarding organ protection. Furthermore, improved organ storage after procurement - such as machine perfusion - can diminish effects of IRI in a clinical setting. Brain death plays a fundamental role in the regulation of molecular markers triggering inflammation and IRI-related tissue damage in liver transplants. Although several treatment options have reached clinical application, to date, the effects of brain death during donor conditioning and organ procurement remain relevant for organ function and survival.

Novel Approaches to Preventing Ischemia-Reperfusion Injury During Liver Transplantation

Ischemia-reperfusion injury (IRI) results in profound allograft damage during liver transplantation. The process of IRI results in adenosine triphosphatase (ATP) depletion, the production of reactive oxygen species, and progressive tissue destruction. This injury process is accelerated on reperfusion in the recipient. Over the last decade an increasing body of literature has identified a complex interplay of molecular and cellular pathways responsible for causing IRI. This article summarizes recent developments, drawing on preclinical and clinical studies, focusing on how the detrimental effects of IRI can be prevented in liver transplantation. We present a balanced overview on how machine preservation technologies, the coagulation system, antioxidants, cytoprotective agents, cytokines, preservation solutions, and the innate and adaptive immune system can be targeted to prevent IRI in liver transplantation.

Reactive Metabolites and AGE-RAGE-Mediated Inflammation in Patients following Liver Transplantation

Recent investigations have indicated that reactive metabolites and AGE-RAGE-mediated inflammation might play an important role in the pathogenesis of ischemia-reperfusion injury in liver transplantation. In this observational clinical study, 150 patients were enrolled following liver transplantation from deceased donors. The occurrence of short-term complications within 10 days of transplantation was documented. Blood samples were collected prior to transplantation, immediately after transplantation, and at consecutive time points, for a total of seven days after transplantation. Plasma levels of methylglyoxal were determined using HPLC, whereas plasma levels of L-arginine, asymmetric dimethylarginine, advanced glycation endproducts-carboxylmethyllysine, soluble receptor for advanced glycation endproducts, and total antioxidant capacity were measured by ELISA. Patients following liver transplantation were shown to suffer from increased RAGE-associated inflammation with an AGE load mainly dependent upon reactive carbonyl species-derived AGEs. In contrast, carboxylmethyllysine-derived AGEs were of a minor importance. As assessed by the ratio of L-arginine/asymmetric dimethylarginine, the bioavailability of nitric oxide was shown to be reduced in hepatic IRI, especially in those patients suffering from perfusion disorders following liver transplantation. For the early identification of patients at high risk of perfusion disorders, the implementation of asymmetric dimethylarginine measurements in routine diagnostics following liver transplantation from deceased donors should be taken into consideration.

The influences of ischemia-reperfusion injury in liver transplantation on intraoperative lung injury and postoperative pulmonary complications

Objective To study the effects of ischemia-reperfusion in liver transplantation on the pathophysiological changes of the lung and mechanisms of lung injury. Methods We studied 23 patients who received liver transplantation at Fuzhou General Hospital of PLA. We cut a small piece of the right lung for pathological study and for L-1β and TNF-a immunohistochemistry studies at 5 minutes after the beginning of operation (Ta), 5 minutes before the portal vein was opened (Tb) and three hours after the new liver was transplanted (Tc). We also collected peripheral blood to study the concentration of IL-1β and TNF-a in the plasma at the beginning of operation (T1), the portal vein 5 minutes before opening, the portal vein (T2) ten minutes after the opening (T3) , and one hour after the new liver was transplanted (T4), three hours after the new liver was transplanted (T5), and 12 hours after operation (T6). Results The cytokines TNF-a and IL-1β in peripheral blood were not obviously increased in the portal vein before it was opened, but were significantly increased after the portal vein was opened. Comparison of T1 and T2 separately with T3, T4 and T5 showed significant differences (P<0. 01). In light and electron microscopy, the structures of the lung tissues were normal at Ta and did not change significantly at Tb. There were significant abnormalities at Tc. The average positive points of TNF-a and IL-1β expressions in the lung tissues at Tc were significantly higher than Ta and Tb(P<0. 01). Conclusion Ischemia-reperfusion in liver transplantation led to a serious systemic inflammatory syndrome,and acute lung injury. TNF-a and IL-1β were involved in acute lung injury. Key words: Liver transplantation; Ischemia-reperfusion; TNF-a; IL-1β; Lung injury

Cell Apoptosis and Fas Gene Expression in Liver and Renal Tissues After Ischemia-Reperfusion Injury in Liver Transplantation

Ischemia-reperfusion (I/R) injury is an important factor in a nonfunctioning liver graft and acute renal failure. Apoptosis is a cell death mechanism in early stages of I/R injury. In the present study, orthotopic liver transplantation (oLT) using a modified double-cuff method was performed in Wistar rats, with sham-operated rats serving as the control group. Rats in the treatment and control groups were sacrificed at 1, 3, 6, 12, and 24 hours after oLT to obtain liver and kidney tissues. Fas protein expression in apoptotic cells at various times was detected at immunohistochemical staining and flow cytometry, and Fas gene expression was detected using the reverse transcriptase polymerase chain reaction. Apoptosis began in liver and renal cells at 1 hour after oLT, peaking at 12 hours. The reverse transcriptase polymerase chain reaction demonstrated Fas gene expression in liver and renal tissues at 1 hour post-oLT, peaking at 12 hours. Changes in the treatment group were significantly greater than in the control group ( P < .05). We conclude that renal cells, like liver cells, undergo apoptosis due to I/R injury after oLT.

The Effects of Cyclooxygenase (COX)-2 Inhibition on Ischemia-Reperfusion Injury in Liver Transplantation

Purpose: Our objective was to evaluate whether COX-2 inhibition with FK3311, a selective cyclooxygenase (COX)-2 inhibitor, improves transplanted liver function. Methods: Inbred male Lewis rats weighing 200–260 g were used. The donor liver was perfused with cold University of Wisconsin (UW) solution and then stored in the same solution at 4°C for 18 hr. After the preservation period, orthotopic liver transplantation was performed. Animals were divided into three groups: the control group; the FK low-dose group (1 mg/kg FK3311 i.v. 20 min before reperfusion); and the FK high-dose group (3 mg/kg FK3311 i.v. 20 min before reperfusion). Survival rate, serum GOT and GPT levels, liver tissue blood flow, and serum thromboxane B2 (TxB2) levels were compared among groups. Results: Survival rate was significantly better (p <. 05) and serum GOT levels 30 min after reperfusion were significantly lower (p <. 05) in the FK high-dose group compared to the other two groups. Four hours after reperfusion, GPT levels and liver tissue flow were significantly (p <. 05) better in the FK high-dose group compared to the control. Both 30 min and 4 hr after reperfusion, serum TxB2 levels were significantly lower in the FK high-dose group compared to the control (p <. 05). Conclusion: COX-2 activity results in deteriorated liver function after I/R injury associated with transplantation, and selective COX-2 inhibition improved liver graft function.

Diannexin, a Novel Annexin V Homodimer, Protects Rat Liver Transplants Against Cold Ischemia‐Reperfusion Injury

Ischemia/reperfusion injury (IRI) remains an important problem in clinical transplantation. Following ischemia, phosphatidylserine (PS) translocates to surfaces of endothelial cells (ECs) and promotes the early attachment of leukocytes/platelets, impairing microvascular blood flow. Diannexin, a 73 KD homodimer of human annexin V, binds to PS, prevents attachment of leukocytes/platelets to EC, and maintains sinusoidal blood flow. This study analyzes whether Diannexin treatment can prevent cold IRI in liver transplantation. Rat livers were stored at 4 degrees C in UW solution for 24 h, and then transplanted orthotopically (OLT) into syngeneic recipients. Diannexin (200 microg/kg) was infused into: (i) donor livers after recovering and before reperfusion, (ii) OLT recipients at reperfusion and day +2. Controls consisted of untreated OLTs. Both Diannexin regimens increased OLT survival from 40% to 100%, depressed sALT levels, and decreased hepatic histological injury. Diannexin treatment decreased TNF-alpha, IL-1beta, IP-10 expression, diminished expression of P-selectin, endothelial ICAM-1, and attenuated OLT infiltration by macrophages, CD4 cells and PMNs. Diannexin increased expression of HO-1/Bcl-2/Bcl-xl, and reduced Caspase-3/TUNEL+ apoptotic cells. Thus, by modulating leukocyte/platelet trafficking and EC activation in OLTs, Diannexin suppressed vascular inflammatory responses and decreased apoptosis. Diannexin deserves further exploration as a novel agent to attenuate IRI, and thereby improve OLT function/increase organ donor pool.

Molecular Characterization of Rat Leukocyte P-Selectin Glycoprotein Ligand-1 and Effect of Its Blockade: Protection from Ischemia-Reperfusion Injury in Liver Transplantation

P-selectin glycoprotein ligand-1 (PSGL-1) mediates the initial tethering of leukocytes to activated platelets and endothelium. We report molecular cloning and characterization of the rat PSGL-1 gene. A neutralizing Ab was generated, and its binding epitope was mapped to the N-terminal binding region of rat PSGL-1. We examined the effects of early PSGL-1 blockade in rat liver models of cold ischemia, followed by ex vivo reperfusion or transplantation (orthotopic liver transplantation (OLT)) using an anti-PSGL-1 Ab with diminished Fc-mediated effector function. In the ex vivo hepatic cold ischemia and reperfusion model, pretreatment with anti-PSGL-1 Ab improved portal venous flow, increased bile production, and decreased hepatocellular damage. Rat pretreatment with anti-PSGL-1 Ab prevented hepatic insult in a model of cold ischemia, followed by OLT, as assessed by 1) decreased hepatocellular damage (serum glutamic oxaloacetic transaminase/glutamic-pyruvic transaminase levels), and ameliorated histological features of ischemia/reperfusion injury, consistent with extended OLT survival; 2) reduced intrahepatic leukocyte infiltration, as evidenced by decreased expression of P-selectin, ED-1, CD3, and OX-62 cells; 3) inhibited expression of proinflammatory cytokine genes (TNF-alpha, IL-1beta, IL-6, IFN-gamma, and IL-2); and 4) prevented hepatic apoptosis accompanied by up-regulation of antiapoptotic Bcl-2/Bcl-xL protective genes. Thus, targeting PSGL-1 with a blocking Ab that has diminished Fc-mediated effector function is a simple and effective strategy that provides the rationale for novel therapeutic approaches to maximize the organ donor pool through the safer use of liver transplants despite prolonged periods of cold ischemia.