Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival times. The efficacy of current systemic therapies for HCC is limited. In this study, we used xenograft tumor models to investigate the use of antibodies that block CD47 and inhibit HCC tumor growth. Immunostaining of tumor tissue and HCC cell lines demonstrated CD47 over-expression in HCC as compared to normal hepatocytes. Macrophage phagocytosis of HCC cells was increased after treatment with CD47 antibodies (CD47mAbs) that block CD47 binding to SIRPα. Further, CD47 blockade inhibited tumor growth in both heterotopic and orthotopic models of HCC, and promoted the migration of macrophages into the tumor mass. Our results demonstrate that targeting CD47 by specific antibodies has potential immunotherapeutic efficacy in human HCC.

UW solution and HTK solution are both used for cold preservation of liver allografts. Although they are about equally effective, their compositions are very different, and they were formulated using different rationales. The authors recently showed an important role for MMPs in liver preservation injury and consequently postulated that these preservation solutions contain cryptic inhibitors of MMP activity. To determine this possibility, the ability of these solutions to inhibit MMP activity was studied. The source of MMP2 and MMP9 was human liver effluents obtained at the time of liver transplantation or commercially available human recombinant MMP2 and MMP9. MMP2 and MMP9 showed gelatinolytic activity at 37ЊC and also at 4ЊC, although activity at 4ЊC was reduced. Activity was inhibited by University of Wisconsin (UW) and Histidine/Tryptophan/ Ketoglutarate (HTK) solutions. Examination of individual ingredients disclosed that reduced glutathione (GSH) and lactobionate in UW solution and histidine in HTK solution were the cryptic inhibitors. HTK solution was a more effective inhibitor than UW solution. GSH inhibited the activity of both enzymes, but was a much more effective inhibitor of MMP9 than MMP2. Oxidized glutathione(GSSG) was a much less effective inhibitor of the enzymes. The inhibitor constants (K i ) of GSH for MMP2 and MMP9 were 34 mol/L and 3 mol/L, respectively. The authors conclude that MMP inhibition is a cryptic property of both commonly used liver preservation solutions and contributes importantly to their action. Furthermore, GSH appears to be an effective inhibitor of gelatinases at concentrations at which it is normally present in extracellular fluid. (HEPATOLOGY 2000;31:1115-1122.)University of Wisconsin (UW) solution became the standard liver preservation solution more than 10 years ago. 1,2 It greatly improved the results of hepatic preservation compared with Eurocollins solution, which was in common use before that time. 1,2 UW solution was formulated with specific ingredients to retard the 4 classical effects of hypothermiacell swelling, impaired energy metabolism, acidosis, and accumulation of precursors of reactive oxygen intermediates. 2 Metalloproteinase inhibition was not a goal of its formulation because the role of metalloproteinases in preservation injury 3 was unknown at the time that UW solution was devised.Bretschneider' s solution, also known as histidine/tryptophan/ketoglutarate solution, or HTK solution 4 is also used for liver preservation, mainly in Germany. HTK solution was originally introduced as a cardioplegic solution and was later adapted to cold preservation of organs. It was formulated to retard acidosis (histidine), to prevent membrane injury (tryptophan), and to provide a substrate for energy metabolism (ketoglutarate). Like UW solution, inhibition of metalloproteinases was not a goal of its formulation.Two recent human studies, 5,6 one of which was a randomized controlled trial, 6 compared effectiveness of UW and HTK solutions. These studies found that the sol...

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Hepatic steatosis continues to present a major challenge in liver transplantation. These organs have been shown to have increased susceptibility to cold ischemia/reperfusion (CIR) injury in comparison with otherwise comparable lean livers; the mechanisms governing this increased susceptibility to CIR injury are not fully understood. Endoplasmic reticulum (ER) stress is an important link between hepatic steatosis, insulin resistance, and metabolic syndrome. In this study, we investigated ER stress signaling and blockade in the mediation of CIR injury in severely steatotic rodent allografts. Steatotic allografts from genetically leptin-resistant rodents had increased ER stress responses and increased markers of hepatocellular injury after liver transplantation into strain-matched lean recipients. ER stress response components were reduced by the chemical chaperone taurine-conjugated ursodeoxycholic acid (TUDCA), and this resulted in an improvement in the allograft injury. TUDCA treatment decreased nuclear factor kappa B activation and the proinflammatory cytokines interleukin-6 and interleukin-1b. However, the predominant response was decreased expression of the ER stress cell death mediator [CCAAT/enhancer-binding protein homologous protein (CHOP)]. Furthermore, activation of inflammation-associated caspase-11 was decreased, and this linked ER stress/CHOP to proinflammatory cytokine production after steatotic liver transplantation. These data confirm ER stress in steatotic allografts and implicate this as a mediating mechanism of inflammation and hepatocyte death in the steatotic liver allograft.

Background Steatotic liver grafts tolerate ischemia-reperfusion (I/R) injury poorly, contributing to increased primary graft nonfunction following transplantation. Activation of nuclear factor kappa-B (NFκB) following I/R injury plays a crucial role in activation of pro-inflammatory responses leading to injury. Methods We evaluated the role of NFκB in steatotic liver injury by using an orthotopic liver transplant (OLT) model in Zucker rats (lean to lean or obese to lean) to define the mechanisms of steatotic liver injury. Obese donors were treated with bortezomib to assess the role of NF-κB in steatotic liver I/R injury. Hepatic levels of NF-κB and pro-inflammatory cytokines were analyzed by ELISA. Serum transaminase levels and histopathological analysis were performed to assess associated graft injury. Results I/R injury in steatotic liver results in significant increases in activation of NF-κB (40%, p<0.003), specifically the p65 subunit following transplantation. Steatotic donor pretreatment with proteasome inhibitor bortezomib (0.1 mg/kg) resulted in significant reduction in levels of activated NF-κB (0.58±0.18 vs. 1.37±0.06 O.D./min/10μg protein, p<0.003). Bortezomib treatment also reduced expression of pro-inflammatory cytokines MIP-2 compared with control treated steatotic and lean liver transplants respectively (106±17.5 vs. 443.3±49.9 vs. 176±10.6 pg/mL, p=0.02), TNF-α (223.8±29.9 vs. 518.5±66.5 vs 264.5±30.1 pg/2μg protein, p=0.003) and IL-1β (6.0±0.91 vs. 19.8±5.2 vs 5±1.7 pg/10μg protein, p= 0.02) along with a significant reduction in ALT levels (715±71 vs 3712.5±437.5 vs 606±286 U/L, p=0.01). Conclusion These results suggest that I/R injury in steatotic liver transplantation are associated with exaggerated activation of NFκB subunit p65, leading to an inflammatory mechanism of reperfusion injury and necrosis. Proteasome inhibition in steatotic liver donor reduces NFκB p65 activation and inflammatory I/R injury, improving transplant outcomes of steatotic grafts in a rat model.

Cold preservation induces the secretion of matrix metalloproteinases (MMPs) by hepatic sinusoidal endothelial cells (SECs). These enzymes are important mediators of cold preservation injury. The purpose of this study was to determine if low temperature caused actin disassembly in SECs and whether actin disassembly was required for secretion of MMPs under these conditions. To establish the basis of interpreting the effect of low temperature, isolated SECs were exposed to cytochalasin B with or without pretreatment with phalloidin. Cytochalasin B produced actin disassembly and resulted in the secretion of MMPs. Both were retarded by phalloidin pretreatment. Low temperature (4ЊC) also induced actin disassembly and MMP secretion and pretreatment with phalloidin again retarded actin disassembly and MMP secretion. Cycloheximide had no effect on these results. Actin disassembly began with 30 minutes of exposure of isolated SECs to cold and reached a final state at 8 hours, at which time no actin stress fibers were visible, and the normally fusiform SECs were fully rounded. Increased MMP activity in the supernatant was also present at 30 minutes and continued to rise sharply in the first hour; thereafter the rate of rise diminished. The study shows that secretion of MMPs during cold preservation is dependent on the induction of actin disassembly by low temperature. The rapid appearance of increased MMP activity after exposure to cold and the studies using cycloheximide indicate that the MMPs originate from preformed MMPs rather than newly synthesized MMPs. (HEPA-TOLOGY 1999;30:169-176.)Preservation injury leads to hepatic failure and the loss of about 8% of transplanted livers in the United States. [1][2][3] Preservation injury also restricts strategies to increase the use of donor organs, for instance, by splitting 1 liver for use in 2 adults. Livers are damaged during low temperature (4°C) preservation by a well-documented injury to the hepatic sinusoidal endothelial cells, 4,5 while hepatocytes are relatively spared. 4 During cold preservation, SECs partially detach from the connective tissue matrix of the sinusoidal wall and become rounded in shape, apparently connected to the matrix only by isolated cords of cytoplasm. 4,6 After preservation, at the moment of reperfusion, recipient leukocytes, platelets, and clotting factors come into contact with the exposed donor connective tissue matrix and hepatocytes, as well as the altered sinusoidal endothelial cells (SECs), in the hepatic sinusoid. This leads to adherence and activation of the platelets 7 and leukocytes 8,9 as well as thrombosis 10 with resultant damage to the allograft. The degree to which these events occur on reperfusion is directly related to the duration of cold preservation in every model.Recently, we offered an explanation for the detachment and rounding of SECs based on pathological activity of matrix metalloproteinases (MMPs). 11 We found that MMPs are present in liver effluents after cold preservation in both man and in the rat and that their acti...

Normothermic extracorporeal liver perfusion (NELP) can decrease ischemia/reperfusion injury to the greatest degree when cold ischemia time is minimized. Warm perfusion of cold-stored livers results in hepatocellular damage, sinusoidal endothelial cell (SEC) dysfunction, and Kupffer cell activation. However, the logistics of organ procurement mandates a period of cold preservation before NELP. The aim of this study was to determine the beneficial effects of gradual rewarming of cold-stored livers by placement on NELP. Three female porcine livers were used for each group. In the immediate NELP group, procured livers were immediately placed on NELP for 8 hours. In the cold NELP group, livers were cold-stored for 4 hours followed by NELP for 4 hours. In rewarming groups, livers were cold-stored for 4 hours, then gradually rewarmed in different durations to 388C and kept on NELP for an additional 4 hours. For comparison purposes, the last 4 hours of NELP runs were considered to be the evaluation phase. Immediate NELP livers had significantly lower concentrations of liver transaminases, hyaluronic acid, and b-galactosidase and had higher bile production compared to the other groups. Rewarming livers had significantly lower concentrations of hyaluronic acid and b-galactosidase compared to the cold NELP livers. In addition, there was a significant decline in international normalized ratio values, improved bile production, reduced biliary epithelial cell damage, and improved cholangiocyte function. Thus, if a NELP machine is not available at the procurement site and livers will need to undergo a period of cold preservation, a gradual rewarming protocol before NELP may greatly reduce damages that are associated with reperfusion. In conclusion, gradual rewarming of cold-preserved livers upon NELP can minimize the hepatocellular damage, Kupffer cell activation, and SEC dysfunction. Liver Transpl 22:333-343, 2016. V C 2015 AASLD.Received July 22, 2015; accepted September 30, 2015.Normothermic extracorporeal liver perfusion (NELP) has created a new paradigm in the liver preservation field. Growing interest in using this system is based on studies that have shown the efficacy and superiority of warm perfusion over standard static cold storage of the liver.1-3 NELP attenuates ischemia/reperfusionAbbreviations: ALP, alkaline phosphatase; ELISA, enzyme-linked immunosorbent assay; H & E, hematoxylin-eosin; HTK, histidine tryptophan ketoglutarate; INR, international normalized ratio; IRI, ischemia/reperfusion injury; LDH, lactate dehydrogenase; NELP, normothermic extracorporeal liver perfusion; SD, standard deviation; SEC, sinusoidal endothelial cell.

I n the past several years, we have examined intracellular events that occur in sinusoidal endothelial cells (SEC) when they are placed in the cold. 1-4 SEC are the main targets of cold preservation injury, 5,6 and understanding the mechanisms by which they are injured might provide an avenue to development of improved preservation solutions. Our studies show that cold causes actin disassembly 2 and that this is associated with rounding of the cells 2 and release of matrix metalloproteases (MMPs) into the supernatants of SEC cell cultures. 1,3 Actin disassembly and MMP secretion lead to activation of the cell surface, as shown by increased expression of von Willebrand factor and increased adhesiveness for platelets. 4 Platelet adhesion is one of the key events that leads to allograft injury on reperfusion. 7,8 To date, the cause of cold-induced actin disassembly is unclear, but there are suggestions that abnormalities of calpain and calcium metabolism are important. There is a sharp rise in intracellular calcium when SECs are placed in the cold in the presence of extracellular calcium. 9 Calpain is a calcium-dependent intracellular protease the activity of which is increased in whole rat livers preserved in the cold 10 and in human allografts that show poor function after reperfusion. 11 Calpain activity has been shown to be associated with remodeling of actin. 12-14 Therefore, we hypothesized that exposure of SEC to cold results in the following sequence: elevated intracellular calcium concentration, increased calpain activity, and actin disassembly.