Liver ischemia-reperfusion injury: From trigger loading to shot firing.

P73 Aims: Ischemia-reperfusion injury is responsible for the morbidity associated with liver surgery under total vascular exclusion or after liver transplantation. To reduce the injury, ischemic preconditioning has been shown to protect some organs such as heart, kidney, and liver against a more prolonged ischemic insult. Recently, mitochondrial KATP (mito KATP) channel rather than surface KATP channels, has emerged as likely mediators of preconditioning in the heart. Therefore, it has been reported that KATP channel opener has an effect on myocardial protection via an effort to inquire into the pharmacological preconditioning action. However, the final effecter molecules of the action remain unclear. It is unclear even whether KATP channel openers can reduce ischemia-reperfusion injury in the liver. The aim of this study is to determine the effect of mito KATP channel opener, nicorandil (SG-75), on ischemia-reperfusion injury in rat liver. Methods: Male Wistar rats were used and anesthetized with urethane. The blood supply to right and middle lobe of the liver was interrupted to 73% ischemia for 45 min, followed by 120 min of reperfusion. SG-75 (3 mg/kg) was orally administered 60 min before hepatic ischemia. Rats were sacrificed at 120 min after reperfusion (IR group) and after reperfusion with SG-75 administration (SG+IR group). Samples of blood and liver tissues were collected at the indicated times. Plasma levels of ALT and LDH were measured for examination of the liver function. Apoptosis was determined by TUNEL staining. The expression of cytochrome c, Bax, Bcl-2 and caspase-3 were analyzed by western blotting. TNF-α levels of plasma and liver were quantified using ELISA. Results: Plasma levels of ALT and LDH in IR group markedly increased to 5588 ± 646 and 28270 ± 3325 (IU / L), respectively. SG-75 significantly decreased their levels by about 60% (p<0.05). TUNEL-positive hepatocytes remarkably increased in about 30% of total hepatocytes in IR group. In SG-75 group, the increase of TUNEL-positive hepatocytes was inhibited to about 10%. Levels of cytochrome c in the corresponding mitochondrial fraction of the liver did not differ between the two groups. Cytochrome c and activated caspase-3 levels in the cytosol increased in IR group and SG-75 inhibited the release of cytochrome c and activation of caspase-3. The expression of Bax and Bcl-2 significantly increased in IR group and was slightly inhibited by administration of SG-75. Hepatic TNF-α levels slightly increased after reperfusion in both groups. Conclusions: These results suggested that its protective effect of SG-75 against hepatic ischemia-reperfusion injury was correlated with the inhibition of mitochondrial cytochrome c release and caspase-3 activation. These findings demonstrate that SG-75 may also become a therapeutic drug for IR-related hepatocellular damage.

Ischemia-reperfusion injury (IRI) represents a major determinant of liver transplantation. IRI-induced graft dysfunction is related to biliary damage, partly due to a loss of bile canaliculi (BC) integrity associated with a dramatic remodeling of actin cytoskeleton. However, the molecular mechanisms associated with these events remain poorly characterized. Using liver biopsies collected during the early phases of organ procurement (ischemia) and transplantation (reperfusion), we characterized the global patterns of expression and phosphorylation of cytoskeleton-related proteins during hepatic IRI. This targeted functional proteomic approach, which combined protein expression pattern profiling and phosphoprotein enrichment followed by mass spectrometry analysis, allowed us to identify IQGAP1, a Cdc42/Rac1 effector, as a potential regulator of actin cytoskeleton remodeling and maintenance of BC integrity. Cell fractionation and immunohistochemistry revealed that IQGAP1 expression and localization were affected upon IRI and related to actin reorganization. Furthermore using an IRI model in human hepatoma cells, we demonstrated that IQGAP1 silencing decreased the basal level of actin polymerization at BC periphery, reflecting a defect in BC structure coincident with reduced cellular resistance to IRI. In summary, this study uncovered new mechanistic insights into the global regulation of IRI-induced cytoskeleton remodeling and led to the identification of IQGAP1 as a regulator of BC structure. IQGAP1 therefore represents a potential target for the design of new organ preservation strategies to improve transplantation outcome.

Neutrophils: a cornerstone of liver ischemia and reperfusion injury

You celebrated 6000 liver transplants at UCLA in 2016. How does one build the largest liver transplant program in the world?FigureRWB: The building of our Liver Transplantation Program at UCLA, was a complex undertaking stimulated by a 20-year-old patient whom I had performed a distal splenorenal shunt for variceal bleeding. Unexpectedly, he developed liver failure 7 days postoperatively and died, before I could transfer him to Dr. Starzl in Pittsburgh for consideration of liver transplantation. As I walked out of the ICU that day, I turned to my friend and hepatology colleague, Dr. Leonard Goldstein and stated “Leonard, we need to start doing liver transplants at UCLA.” In late 1982, I told Dr. William P. Longmire, Jr., a renowned liver surgeon and Chairman at UCLA, that I would like to start a liver transplant program. He was supportive, and I assembled a team and initiated a program of porcine orthotopic liver transplantation. We performed over 50 transplants using venous-venous bypass, with excellent success. I then visited Dr. Starzl in Pittsburgh to observe clinical liver transplantation and participated in about 6 cases. Over the next several months, we put together our multidisciplinary team at UCLA despite some skepticism from hospital administration. On February 1, 1984, we performed our first liver transplant on a recipient with a hepatic schwannoma and used venous venous bypass. The patient required 17 units of blood and was discharged on postoperative day 17. We performed our 100th liver transplant at UCLA in November 1986, and reported our experience the following year at the American Surgical Association. In my closing remarks, I recognized Dr. Starzl’s contributions: “how well I remember the multiple phone conversations on our first few transplants, in which I sought your advice, encouragement and leadership and I am very grateful for that.” After our first 100 liver transplants, our program continued to grow rapidly since we were one of the first programs in the western part of the United States, and encompassed the entire spectrum of pediatric and adult liver transplantation including the sickest patients. Our team performed our 5000th liver transplant on September 9, 2010, and our 6000th on June 30, 2016, making our program one of the largest worldwide. None of this could have happened without the unfailing support of our totally dedicated multidisciplinary team of surgical and medical specialists, nurse coordinators, hospital leadership, administrators, organ procurement agency, and the supportive generosity of donor families. More than 6000 liver transplants would not go by without remembering very special cases. What is your most memorable surgery and patient? RWB: To be honest, all of my patients are memorable. Certainly, those who did not survive have influenced me in ways that resulted in improving our patient and donor selection, operative techniques and postoperative management. If we look for instance at our pediatric patients there has been a significant improvement in 15 year graft survival from 51% from 1984 to 2000 to 72% from 2001 to 2017. Although all of my patients are memorable, there is a very special 1-year-old child, who I transplanted on August 8, 1984, our fifth transplant, with a giant hepatic hemangioendothelioma who is now 34 years old, married, and living a wonderful life. She is one of over 1000 children that we have transplanted in our program. In addition to excellent clinical outcomes, you have a unique collection of clinical data. How did you built your database and what have you learnt and brought back to clinical application? RWB: From the inception of our program, each patient evaluated for liver transplantation has been registered into an IRB sanctioned transplant database, with a comprehensive list of recipient, donor, and perioperative variables maintained prospectively. Over the years, our transplant surgeons have played the leading role in extracting additional clinical, laboratory, radiologic, and pathologic information from the medical records, resulting in the continuous growth and enrichment of the database which is updated regularly. This robust research database has been integral to our research productivity over the years. With greater than 800 publications in peer-reviewed journals, the UCLA transplant program has made significant contributions to the field of liver transplantation, with leading roles in numerous randomized-controlled trials that led to the current standard of care in immunosuppression (tacrolimus), and fungal, viral, and PCP prophylaxis following LT. Furthermore, we have contributed important innovations in surgical techniques such as in situ split-liver transplantation, as well as innumerable reports of clinical outcomes examining salient issues pertaining to donor allocation, use of extended-criteria donor allografts, and transplantation for malignancies. Our clinical research program is integrated with our robust basic science and translational program focusing on hepatic ischemia/reperfusion injury, leading to several clinical trials in human liver transplantation. You have mentored many transplant surgeons who went on to take very successful leadership roles. What is the secret of your mentoring style? RWB: I truly believe that one of the most important components of my career has been my commitment to training and mentoring future leaders in transplantation. This is a multifaceted commitment, and as stated by Gary Burnison, CEO of Korn Ferry International, “to lead is to be all in, transparent and accessible, calm in the face of upset and even crisis, and always mindful that you are a steward of something bigger than yourself.” Transplantation is certainly a discipline which demands all of the above. Additionally, to be successful in training our future leaders, you must demonstrate vision, self-direction, courage to take on complex cases, and most importantly to always embody honesty and integrity. Finally, genuine, personal interaction is essential. Despite my administrative role as Chairman of our Department, I always set time aside for personal interaction and the mentoring of medical students, residents and fellows, which includes clinical rounding, one-on-one meetings, and hosting a monthly Journal Club at my house for the last 30 years. The LA Times had an interview, now almost 2 decades back in which they featured you and your efforts in finding novel ways to keep up with the demand for liver transplantation. All those attempts, at the time seemed unable to keep up with havoc caused by hepatitis C. Today, new and effective antivirals have changed the game. How have the new antivirals changed liver transplantation? RWB: Hepatitis C was clearly the most common indication for liver transplantation in most centers in the United States until the advent of effective antiviral agents over the past couple of years. However, even patients who have cleared the virus may still require liver replacement due to failing liver function. In these cases, the results of liver transplant are vastly improved due to the lack of HCV recurrence. HCV is diminishing as an indication for liver transplant, and we now have a new leader in the queue, which is nonalcoholic steatohepatitis (NASH). In many cases, these patients are more complex, more technically demanding and have additional co-morbidity. We are pushing to establish a trial to determine if sleeve gastrectomy performed with liver transplantation will improve the outcomes of these difficult patients. There has been a long debate on the timing for patients with end-stage alcohol toxic liver disease. Is it safe to transplant those patients without a minimum time of documented abstinence? RWB: Liver transplantation for alcoholic hepatitis is a very controversial topic, due to the high rate of recidivism and the limited donor pool. However, there is more data coming out which shows that early liver transplantation for severe alcoholic hepatitis in selected patients can provide very good short-term survival and equivalent rates of relapse as seen in patients who have 6 months of abstinence pretransplant. One of my former fellows, Dr. Andrew Cameron, Chief of Liver Transplantation at Johns Hopkins University, recently published the results of a 3-year pilot program comparing patients with alcoholic hepatitis after first liver decompensation versus those with the same condition that had 6 months of abstinence. The survival and incidence of alcohol relapse was the same in both groups. In an editorial that I authored for this article, I concluded that the dramatic improvement in survival in those transplanted early and the equivalent recidivism rates compared to those transplanted after 6 months sobriety justifies this approach and careful consideration should be given to implementing this policy with close scrutiny. You list more than 700 publications in PubMed. What do you consider your most important scientific contribution? Together with Dr. Jerzy Kupiec-Weglinski you have explored many novel mechanistic and therapeutic avenues addressing ischemia/reperfusion injury. What of those efforts have been or about to be translated into clinical application? RWB: I have been intensively involved in both clinical and basic science research since I was a medical student at Tulane, where I obtained my MD and MS degrees. My masters degree thesis was “The Cytological Localization of Erythropoietin Using the Fluorescent Antibody Technique”. Upon obtaining my PhD in 1975, I published one of the first articles demonstrating that steroid therapy was successful in blocking ischemia reperfusion injury in ischemic hearts. Since founding the liver transplant program in 1984, my basic research program has been focused on the prevention of ischemia reperfusion injury (IRI) of the liver. I have been continually funded from the NIH and other peer-reviewed granting agencies since 1981. In 1997, I recruited Jerzy Kupiec-Weglinski, MD, PhD, from Harvard University to lead the basic science thrust of our laboratory. I have worked very closely with Dr. Kupiec-Weglinski to specifically identify the mechanisms of (IRI) and to develop treatment modalities to prevent the injury. Indeed, our “bench-to-bedside” collaborative research on the innate—adaptive immune interface in liver transplant recipients has been recently awarded a 5-year Program Project Grant from the NIH. As there are less than 10 program project grants in the country funded by the NIH that are related to organ transplantation, this is quite an achievement. The focus of the research on liver ischemia reperfusion injury (IRI) is both basic and translational since IRI contributes to poor graft function after transplantation. Minimizing the adverse effects of IRI could increase the number of patients that may successfully undergo liver transplantation. Our research has involved studying the platelet leukocyte endothelial cell interactions which play a central role in IRI. We were the first group to document that inhibition of P-Selectin activation by blocking P-Selectin glycoprotein ligand-1 was highly successful in increasing survival in marginal liver grafts after transplantation. I have been the principal investigator of these studies since they were initiated in the mid-1990s and currently these have been expanded to the clinical arena with Phase II clinical trials utilizing P-Selectin/PSGL-1 blockade in both kidney and liver transplantation. In 2012, I was the lead author of a randomized placebo controlled phase II clinical trial comparing placebo versus selectin blockade in a series of 47 patients undergoing liver transplantation. Selectin blockade proved to be nontoxic and improved graft survival, liver function tests and biomarkers of inhibition of IRI. This study is the stimulus for a multicenter trial investigating selectin blockade as a mechanism to improve liver graft function and has applicability to other organ transplants. In addition to this basic science research, I have been the senior author on numerous seminal clinical articles which have served as benchmarks in the treatment of liver transplant patients in many areas of clinical focus including: immunosuppression, perioperative viral and fungal prophylaxis, technical modifications, use of extended criteria donors, management of infants undergoing liver transplantation, hepatocellular carcinoma, living donor organ donation, split liver transplantation, combined kidney - liver transplants, multivisceral transplants, and transplantation of patients with the highest Model for End-stage Liver Disease score, which is used to allocate organs. Many of these accomplishments were supported by NIH and other peer-reviewed funding. Looking into the future: what do you see as the main challenges for liver transplantation in the 10 years? RWB: There are indeed numerous challenges for liver transplantation that we will encounter over the next 10 years. Today, liver transplantation is considered the gold standard for treatment of patients with end-stage liver disease. However, new improved treatment strategies, as we now have for HCV and HBV, and in the possible near future for hepatocellular carcinoma will surely decrease the need for liver replacement. In certain metabolic diseases, cellular transplantation may become very effective as the preferred treatment over whole organs. Life-long immunosuppression definitely has its drawbacks. However, once our ability to induce tolerance improves, immunosuppressive drug therapy will be minimized. Furthermore, the new avenues of research such as blockade of ischemia reperfusion injury and the resuscitation of marginal grafts with novel preservation concepts will significantly increase the organ donor pool. Your energy does not seem to stop outside of the hospital walls. There are rumors that a unique collection of Italian sport cars share your home address. Moreover, you are an avid runner and have finished the new your city marathon twice. What do you enjoy outside the operating room? RWB: My career would not have developed were it not for the incredible, selfless, and loving support of my family: my wife of 50 years, JoAnn, our 2 daughters, Amber and Ashley, and my 4 grandsons. One of my passions is indeed automobiles, perhaps a genetic trait inherited from my father, who was a car dealer when I was growing up. I worked in his dealership as a teenager and attended many races including the 12 hours of Sebring, the Monte Carlo Grand Prix, and the Indianapolis 500. I personally raced in the Mille Miglia 1000-mile race in Italy 3 times. I have been playing tennis since I was a medical student, and still exercise daily with a morning or evening run depending on my OR and administrative schedule. My wife, JoAnn is an art enthusiast, collector, and docent for the Los Angeles Museum of Art, and I have enjoyed and benefited from her expertise and passion in this area for many years.

The Ninj1/Dusp1 Axis Contributes to Liver Ischemia Reperfusion Injury by Regulating Macrophage Activation and Neutrophil Infiltration

The protective effects of fibroblast growth factor 10 against hepatic ischemia-reperfusion injury in mice

Liver transplantation is the only effective method for end-stage liver disease; however, liver ischemia reperfusion injury (IRI) seriously affects donor liver function after liver transplantation. IRI is a pathophysiological process in which organ damage is aggravated after the blood flow and oxygen supply of ischemic organ tissues are restored. It combines the two stages of hypoxic cell stress triggered by ischemia and inflammation-mediated reperfusion injury. Herein, we studied the protective effect and mechanism of the anti-T cell Ig and mucin domain (TIM1) monoclonal antibody, RMT1-10, on hepatic cell injury induced by IRI. First, a liver IRI model was established in vivo. HE, TEM, and Tunel were used to detect liver tissue injury, changes in the liver ultrastructure and liver cell apoptosis, respectively. ELISA were performed to determine the levels of ALT, AST, MDA, GSH, and related inflammatory factors. We found that RMT1-10 could significantly reduce liver injury. Flow cytometry results showed that the number of TIM1+ regulatory B cells (Bregs) in the IRI liver increased briefly, while pretreatment with RMT1-10 could increase the number of TIM1+ Bregs and interleukin-10 (IL-10) secretion in liver IRI model mice, thus playing a protective role in liver reperfusion. When Anti-CD20 was used to remove B cells, RMT1-10 had a reduced effect on liver IRI. Previous data showed that the number of T helper 1 cells (Th1:CD4+; CD8+) increased significantly after IRI. RMT1-10 inhibited Th1 cells; however, it significantly activated regulatory T cells. Sequencing analysis showed that RMT1-10 could significantly downregulate the expression of nuclear factor-kappa B (NF-κB) pathway-related genes induced by IRI. These results suggested that RMT1-10 could promote the maturation of B cells through an atypical NF-κB pathway, thereby increasing the number of TIM1+ Bregs and associated IL-10 secretion to regulate the inflammatory response, thereby protecting against liver IRI.

Exacerbated ischemia-reperfusion injury in fatty livers is mediated by lipid peroxidation stress and ferroptosis

Liver disease causing end organ failure is a growing cause of mortality. In most cases, the only therapy is liver transplantation. However, liver transplantation is a complex undertaking and its success is dependent on a number of factors. In particular, liver transplantation is subject to the risks of ischaemia-reperfusion injury (IRI). Liver IRI has significant effects on the function of a liver after transplantation. The cellular and molecular mechanisms governing IRI in liver transplantation are numerous. They involve multiple cells types such as liver sinusoidal endothelial cells, hepatocytes, Kupffer cells, neutrophils and platelets acting via an interconnected network of molecular pathways such as activation of toll-like receptor signalling, alterations in micro-RNA expression, production of ROS, regulation of autophagy and activation of hypoxia-inducible factors. Interestingly, the cellular and molecular events in liver IRI can be correlated with clinical risk factors for IRI in liver transplantation such as donor organ steatosis, ischaemic times, donor age, and donor and recipient coagulopathy. Thus, understanding the relationship of the clinical risk factors for liver IRI to the cellular and molecular mechanisms that govern it is critical to higher levels of success after liver transplantation. This in turn will help in the discovery of therapeutics for IRI in liver transplantation - a process that will lead to improved outcomes for patients suffering from end-stage liver disease.

Ischemia reperfusion injury (IRI) during liver transplantation increases morbidity and contributes to allograft dysfunction. There are no therapeutic strategies to mitigate IRI. We examined a novel hypothesis: caspase 1 and caspase 11 serve as danger-associated molecular pattern (DAMPs) sensors in IRI. By performing microarray analysis and using caspase 1/caspase 11 double-knockout (Casp DKO) mice, we show that the canonical and non-canonical inflammasome regulators are upregulated in mouse liver IRI. Ischemic pre (IPC)- and post-conditioning (IPO) induce upregulation of the canonical and non-canonical inflammasome regulators. Trained immunity (TI) regulators are upregulated in IPC and IPO. Furthermore, caspase 1 is activated during liver IRI, and Casp DKO attenuates liver IRI. Casp DKO maintained normal liver histology via decreased DNA damage. Finally, the decreased TUNEL assay-detected DNA damage is the underlying histopathological and molecular mechanisms of attenuated liver pyroptosis and IRI. In summary, liver IRI induces the upregulation of canonical and non-canonical inflammasomes and TI enzyme pathways. Casp DKO attenuate liver IRI. Development of novel therapeutics targeting caspase 1/caspase 11 and TI may help mitigate injury secondary to IRI. Our findings have provided novel insights on the roles of caspase 1, caspase 11, and inflammasome in sensing IRI derived DAMPs and TI-promoted IRI-induced liver injury.

Although mechanisms of immune activation against liver ischemia reperfusion (IR) injury (IRI) have been studied extensively, questions regarding liver-resident macrophages, that is, Kupffer cells (KCs), remain controversial. Recent progress in the biology of tissue-resident macrophages implicates homeostatic functions of KCs. This study aims to dissect responses and functions of KCs in liver IRI. In a murine liver partial warm ischemia model, we analyzed liver-resident versus infiltrating macrophages by FACS and immunofluorescence staining. Our data showed that liver immune activation by IR was associated with not only infiltrations/activations of peripheral macrophages, but also necrotic depletion of KCs. Inhibition of receptor-interacting protein 1 (RIP1) by necrostatin-1s protected KCs from ischemia-induced depletion, resulting in the reduction of macrophage infiltration, suppression of proinflammatory immune activation, and protection of livers from IRI. The depletion of KCs by clodronate liposomes abrogated the effect of necrostatin-1s. Additionally, liver reconstitutions with KCs postischemia exerted anti-inflammatory/cytoprotective effects against IRI. These results reveal a unique response of KCs against liver IR, that is, RIP1-dependent necrosis, which constitutes a novel mechanism of liver inflammatory immune activation in the pathogenesis of liver IRI.

Previous studies have revealed the activation of neutral sphingomyelinase (N-SMase)/ceramide pathway in hepatic tissue following warm liver ischemia reperfusion (IR) injury. Excessive ceramide accumulation is known to potentiate apoptotic stimuli and a link between apoptosis and endoplasmic reticulum (ER) stress has been established in hepatic IR injury. Thus, this study determined the role of selective N-SMase inhibition on ER stress and apoptotic markers in a rat model of liver IR injury. Selective N-SMase inhibitor was administered via intraperitoneal injections. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60 min, followed by 60 min reperfusion. Levels of sphingmyelin and ceramide in liver tissue were determined by an optimized multiple reactions monitoring (MRM) method using ultrafast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Spingomyelin levels were significantly increased in all IR groups compared with controls. Treatment with a specific N-SMase inhibitor significantly decreased all measured ceramides in IR injury. A significant increase was observed in ER stress markers C/EBP-homologous protein (CHOP) and 78 kDa glucose-regulated protein (GRP78) in IR injury, which was not significantly altered by N-SMase inhibition. Inhibition of N-SMase caused a significant reduction in phospho-NF-kB levels, hepatic TUNEL staining, cytosolic cytochrome c, and caspase-3, -8, and -9 activities which were significantly increased in IR injury. Data herein confirm the role of ceramide in increased apoptotic cell death and highlight the protective effect of N-SMase inhibition in down-regulation of apoptotic stimuli responses occurring in hepatic IR injury.

Liver ischemia and reperfusion injury (IRI) remains a serious clinical problem affecting liver transplantation outcomes. IRI causes up to 10% of early organ failure and predisposes to chronic rejection. Cyclooxygenase-2 (COX-2) is involved in different liver diseases, but the significance of COX-2 in IRI is a matter of controversy. This study was designed to elucidate the role of COX-2 induction in hepatocytes against liver IRI. In the present work, hepatocyte-specific COX-2 transgenic mice (hCOX-2-Tg) and their wild-type (Wt) littermates were subjected to IRI. hCOX-2-Tg mice exhibited lower grades of necrosis and inflammation than Wt mice, in part by reduced hepatic recruitment and infiltration of neutrophils, with a concomitant decrease in serum levels of proinflammatory cytokines. Moreover, hCOX-2-Tg mice showed a significant attenuation of the IRI-induced increase in oxidative stress and hepatic apoptosis, an increase in autophagic flux, and a decrease in endoplasmic reticulum stress compared to Wt mice. Interestingly, ischemic preconditioning of Wt mice resembles the beneficial effects observed in hCOX-2-Tg mice against IRI due to a preconditioning-derived increase in endogenous COX-2, which is mainly localized in hepatocytes. Furthermore, measurement of prostaglandin E2 (PGE2 ) levels in plasma from patients who underwent liver transplantation revealed a significantly positive correlation of PGE2 levels and graft function and an inverse correlation with the time of ischemia. Conclusion: These data support the view of a protective effect of hepatic COX-2 induction and the consequent rise of derived prostaglandins against IRI.

Objective To investigate the relationship between the degree of ischemia reperfusion injury (IRI) of donor liver and the expression of Yes-associated protein (YAP) or the recurrence of hepatocellular carcinoma (HCC) after liver transplantation (LT). Methods The clinical date of 69 patients undergoing LT for HCC were retrospectively analyzed and the samples of the donor liver were collected to detect the degree of IRI and the expression of YAP in donor livers. Factors might influence the postoperative disease-free survival (DFS) after LT were analyzed using the Coxregression model. The survival curve was drawn by Kaplan-Meier method. Results The slight ischemia reperfusion injury of donor livers were 58% (40/69), the positive expressions of YAP were 46.4% (32/69). The expression of YAP in donor liver was related to the degree of IRI (χ2=7.369, P=0.007). The result of multivariate analysis showed that the degree of IRI of donor livers, American Joint Committee on cancer (AJCC) stages Ⅲ were independent risk factors influencing tumor recurrence after LT. Conclusion The degree of IRI of donor liver is an independent risk factor for recurrence of HCC after LT. The degree of IRI of donor livers can affect the expression of YAP. Enhancement of YAP expression may be an important mechanism of HCC recurrence after LT with moderate IRI in donor liver. Key words: Liver transplantation; Recurrence of liver cancer; Yes-assoclated protein; Ischemia reperfusion injury; Tumor free survival

We have previously shown that 2-acetylcyclopentanone (2-ACP), an enolate-forming 1,3-dicarbonyl compound, provides protection in cell culture and animal models of oxidative stress. The pathophysiology of ischemia-reperfusion injury (IRI) involves oxidative stress, and, therefore, we determined the ability of 2-ACP to prevent this injury in a rat liver model. IRI was induced by clamping the portal vasculature for 45 minutes (ischemia phase), followed by recirculation for 180 minutes (reperfusion phase). This sequence was associated with substantial derangement of plasma liver enzyme activities, histopathological indices, and markers of oxidative stress. The 2-ACP (0.80-2.40 mmol/kg), administered by intraperitoneal injection 10 minutes prior to reperfusion, provided dose-dependent cytoprotection, as indicated by normalization of the IRI-altered liver histologic and biochemical parameters. The 2-ACP (2.40 mmol/kg) was also hepatoprotective when injected before clamping the circulation (ischemia phase). In contrast, an equimolar dose of N-acetylcysteine (2.40 mmol/kg) was not hepatoprotective when administered prior to reperfusion. Our studies to date suggest that during reperfusion the enolate nucleophile of 2-ACP limits the consequences of mitochondrial-based oxidative stress through scavenging unsaturated aldehyde electrophiles (e.g., acrolein) and chelation of metal ions that catalyze the free radical-generating Fenton reaction. The ability of 2-ACP to reduce IRI when injected prior to ischemia most likely reflects the short duration of this experimental phase (45 minutes) and favorable pharmacokinetics that maintain effective 2-ACP liver concentrations during subsequent reperfusion. These results provide evidence that 2-ACP or an analog might be useful in treating IRI and other conditions that have oxidative stress as a common molecular etiology.