Markus Selzner

Apoptosis of sinusoidal endothelial cells occurs during liver preservation injury by a caspase-dependent mechanism

BACKGROUND Cold ischemia/warm reperfusion (CI/WR) liver injury remains a problem in liver transplants. Sinusoidal endothelial cells (SEC) are a target of CI/WR injury, during which they undergo apoptosis. Because caspase proteases have been implicated in apoptosis, our aim was to determine whether liver CI/WR injury induces a caspase-dependent apoptosis of SEC. METHODS Rat livers were stored in the University of Wisconsin (UW) solution for 24 hr at 4 degrees C and reperfused for 1 hr at 37 degrees C in vitro. Apoptosis was quantitated using the TUNEL assay, and caspase 3 activation determined by immunohistochemical analysis. Rat liver orthotopic liver transplants (OLT) were also performed using livers stored for 30 hr. RESULTS Terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) positive hepatocytes were rare and did not increase during CI/WR injury. In contrast, TUNEL positive SEC increased 6-fold after reperfusion of livers stored under cold ischemic conditions, compared with controls or livers stored but not reperfused. Immunohistochemical analysis demonstrated active caspase 3 only in endothelial cells after CI/WR injury. When IDN-1965, a caspase inhibitor, was given i.v. to the donor animal and added to UW solution and the reperfusion media, TUNEL positive endothelial cells were reduced 63+/-11% (P<0.05). Similarly, the duration of survival after OLT was significantly increased in the presence of the inhibitor. CONCLUSION During liver CI/WR injury: 1) selective apoptosis of endothelial cells occurs; 2) caspase 3 is activated only in endothelial cells; and 3) a caspase inhibitor reduces endothelial cell apoptosis and prolongs animal survival after OLT. The pharmacologic use of caspase inhibitors could prove useful in clinical transplantation.

Ischemic preconditioning protects the steatotic mouse liver against reperfusion injury: an ATP dependent mechanism.

BACKGROUND/AIMS Hepatic steatosis is a major risk factor for liver surgery and transplantation. The increased susceptibility of fatty livers to ischemic injury is associated with a necrotic form of cell death as opposed to apoptosis in lean animals, and is possibly related to low contents of ATP. Ischemic preconditioning, a brief period of ischemia prior to a prolonged period, protects the lean liver against ischemia through anti-apoptotic properties. We evaluated whether ischemic preconditioning also confers protection in the fatty liver and whether it protects against the ATP loss. METHODS The effect of ischemic preconditioning was tested in steatotic and lean livers subjected to 75 min of ischemia and 4 or 24 h of reperfusion. Tissue ATP contents were assessed at various times, and a model of low hepatic ATP contents (starvation) was studied to assess the type of injury following ischemia and the effects of preconditioning. RESULTS Ischemic preconditioning protected steatotic livers against massive necrosis. ATP levels were significantly higher before and after reperfusion in liver subjected to preconditioning when compared to controls. Liver with low baseline ATP levels (starvation) were also associated with necrosis, and were protected by ischemic preconditioning. CONCLUSIONS Ischemic preconditioning mainly protects the fatty liver against necrosis possibly through preservation and restoration of tissue ATP contents.

Increased ischemic injury in old mouse liver: An ATP-dependent mechanism

Although livers exhibit only minimal morphologic changes with age, how older livers tolerate pathologic conditions such as normothermic ischemia is unknown. Young 6‐week‐old mice and old 60‐week‐old mice underwent 60 minutes of hepatic ischemia and various periods of reperfusion. Markers of hepatocyte injury, hepatic energy content, and mitochondrial function were determined. Ischemic preconditioning and glucose injection were evaluated as protective strategies against reperfusion injury in old mice. Reperfusion injury was far worse in old mice compared with mice in the young control group. Ischemic preconditioning was highly protective against reperfusion injury in young but not in old mice. Older livers had dramatically reduced adenosine triphosphate (ATP) levels and glycogen contents. The low intrahepatic energy level in old mice was associated with a reduced mitochondrial ATP production. Preoperative injection of glucose restored the intrahepatic ATP content and protected against reperfusion injury. Furthermore, glucose injection restored the protective effect of ischemic preconditioning, resulting in additive protection when both strategies were combined. Aging of the liver is associated with mitochondrial dysfunction and decreased intrahepatic energy content, resulting in poorer tolerance against ischemic injury. Improving intrahepatic ATP levels in old livers by glucose injection protects the old liver against ischemic injury and restores the protective effects of ischemic preconditioning. Liver Transpl 13:382–390, 2007.

Transgenic mice overexpressing human Bcl-2 are resistant to hepatic ischemia and reperfusion

BACKGROUND/AIMS Apoptosis is a key mechanism of reperfusion injury in the ischemic liver. The apoptotic pathway is highly regulated by anti-apoptotic factors, such as Bcl-2. We evaluated the effect of Bcl-2 overexpression on apoptosis and the activation of the apoptotic cascade after hepatic ischemia and reperfusion. METHODS Ninety minutes of ischemia and reperfusion was performed in Bcl-2 transgenic and non-transgenic mice. Bcl-2 overexpression was determined by immunohistochemistry and Western blot. Liver injury was determined by aspartate aminotransferase (AST), Tunel test and the activation of the apoptotic cascade and animal survival. RESULTS Bcl-2 overexpression was present in all hepatocytes and non-parenchymal liver cells in transgenic mice. Bcl-2 overexpression resulted in significant decreased AST levels after ischemic injury, and complete inhibition of apoptosis. After 90 min of total hepatic ischemia all control mice died, while four transgenic mice survived permanently. Bcl-2 overexpression was associated with inhibition of caspase 3 activation after reperfusion and increased baseline levels of cytoplasmic cytochrome c, caspase 3, and a reduction of Bcl-x(L) production. CONCLUSIONS Bcl-2 overexpression protects against ischemic injury by inhibiting apoptosis. Extensive overproduction of Bcl-2 is associated with a compensatory increase of baseline levels of cytoplasmic cytochrome c and caspase 3, and a deletion of Bcl-x(L).

Downstaging colorectal liver metastases by concomitant unilateral portal vein ligation and selective intra-arterial chemotherapy.

Although selective intrahepatic arterial chemotherapy successfully downstaged irresectable colorectal liver metastases in a previous study, curative resection was rarely possible, as the remnant healthy liver volume was inadequate. This pilot study evaluated the efficacy of concomitant unilateral portal vein ligation and selective intrahepatic arterial chemotherapy in downstaging such tumours.

Water induces autocrine stimulation of tumor cell killing through ATP release and P2 receptor binding

AbstractAlthough exposure of cells to extreme hypotonic stress appears to be a purely experimental set up, it has found an application in clinical routine. For years, surgeons have washed the abdominal cavity with distilled water to lyse isolated cancer cells left after surgery. No data are available supporting this practice or evaluating the potential mechanisms of cell injury under these circumstances. Recent evidence indicates that increases in cell volume stimulate release of adenosine triphosphate and autocrine stimulation of purinergic (P2) receptors in the plasma membrane of certain epithelial cell types. Under physiological conditions, purigenic stimulation can contribute to cell volume recovery through activation of solute efflux. In addition, adenosine triphosphate-P2 receptor binding might trigger other mechanisms affecting cell viability after profound hypotonic stress. This study demonstrates a novel pathway of cell death by apoptosis in human colon cancer cells following a short hypotonic stress. This pathway is induced by transitory cell swelling which leads to extracellular release of adenosine triphosphate (ATP) and specific binding of ATP to P2 receptors (probably P2X7). Extracellular ATP induced activation of caspases 3 and 8, annexin V, release of cytochrome c, and eventually cell death. The effect of ATP can be blocked by addition of (i) apyrase to hydrolyse extracellular ATP and (ii) suramin, a P2 receptor antagonist. Finally, (iii) gadolinium pretreatment, a blocker of ATP release, reduces sensitivity of the cells to hypotonic stress. The adenosine triphosphate-P2 receptor cell death pathway suggests that autocrine/paracrine signaling may contribute to regulation of viability in certain cancer cells disclosed with this pathway.

Liver regeneration after adult living donor and deceased donor split-liver transplants

To the Editors: The article by Humar et al. in the March 2004 issue of Liver Transplantation is interesting in its comparison of the degree of liver regeneration, as measured by computed tomography volumetry, between living donors, living donor right-lobe recipients and split-liver recipients.1 Their results show a significantly higher percentage increase in liver volume in left-lobe split-liver recipients (119.7%; P .0006), in right-lobe split-liver recipients (113.6%; P .01), and in right-lobe living donor recipients (103.9%; P .0002) as compared to right-lobe living donors whose remnant liver volume had attained 78.6% of its ideal liver volume at 3 months postoperatively.1 In a rat model of partial liver graft transplantation, we recently published results showing an enhanced regenerative response associated with short ( 1 hr) periods of cold ischemia as evidenced by a significantly increased number of proliferating cell nuclear antigen and bromodeoxuridine stained hepatocytes in recipients of partial (30%) liver grafts as compared to control animals that underwent a 70% hepatectomy without transplantation.2 This effect was lost when the graft cold preservation time was prolonged to more than 10 hours and was at that point also associated with reduced survival. This finding seems to concord well with the results given in the article by Humar et al. in the split-liver group, where a relatively short cold ischemia, as in the left-lobe recipients,was associated with a higher regenerative response measured by computed tomography volumetry at 3 months.1 In the living donor recipients,however, thecold ischemia timeseems inaccurate, as it is listed as 0 hours, which does not take into account the time required to at least flush the harvested hemiliver with cold preservation solution prior to implantation, and it seems to indicate that no back-table vascular reconstruction was undertaken in any of the cases studied. The presence of higher regeneration seen in the split-liver group as compared to the living donor recipients does, however, indicate that theremaybeanoptimalcold ischemia time below or above which the triggering stimulus for hepatic regeneration is lost. The process of liver regeneration is complex and dependant on a multitude of factors, such as cytokine pathways, transcriptional regulation, andpatternsof geneactivation.3 In our model, a short period of cold ischemia was also associated with a higher production of interleukin 6 and tumor necrosis factor compared to hepatectomy controls, and a prolonged cold ischemia ( 10 hr) resulted in a significant decrease in both proinflammatory cytokines with a significant reduction in the regenerative response.2 Administration of recombinant-interleukin6 reversed this negative effect of prolonged preservation and resulted in an improved regeneration with a significant increase in hepatocyte proliferating cell nuclear antigen stainingwhencompared toanimals that receivedpartial liver transplants subjectedto10hoursofcold ischemiabut without recombinantinterleukin-6 administration.2 Humar et al. looked at a number of clinical parameters shown to play a role in liver regeneration, including donor age, mean graft weight, and recipient graft-to-body-weight ratio.1,3–5 It is difficult, however, to reach a definite conclusion on the impact of these various factors on hepatic regeneration, including cold ischemia time, in view of the small sample size studied in each group. More detailed clinical studies looking at both clinical and molecular factors will be required in the future as partial liver grafts continue to be used. The article by Humar et al. does provide interesting preliminary data, which should generate further work in this field.

Gastroesophageal Devascularization: Sugiura Type Procedures

To improve the effect of Walker’s simple esophageal transection, Sugiura and colleagues refined the technique of esophageal transection, adding the extensive paraesophageal devascularization via abdominal and thoracic incisions. The resulting procedure has been known as the Sugiura procedure. Three different approaches will be described including a spleen-preserving procedure as well as a procedure with an abdominal approach only (i. e., without thoracotomy).

Ischämische Präkonditionierung: Eine neue Strategie zur Verhinderung von Reperfusionsschäden in der Fettleber

Hepatic steatosis is associated with a reduced tolerance against ischemia/reperfusion injury resulting in an increased mortality after liver resection, shock, and trauma. Ischemic preconditioning (a short time of ischemia prior to a sustained ischemic insult) has recently been described as a protective strategy against reperfusion injury in normal livers. We hypothesized that ischemic injury can be prevented by ischemic preconditioning in steatotic livers. Methods: Steatosis was induced by a choline deficient diet in mice. 75 min ischemia of the median and left liver lobes (70%) was performed in fatty and lean mice. In addition, another group of fatty mice was treated with 10 min ischemia and 15 min reperfusion (ischemic preconditioning) prior to 75 min ischemia. Liver injury was determined by AST release. Hepatocyte apoptosis was evaluated by the TUNEL test and caspase 3 activity. Necrosis was quantified by HE p < 0.001). Ischemic preconditioning reduced the AST values of the fatty mice to levels even lower than in lean animals (8300 U/L). After 24 h reperfusion lean animals (5360 U/L) and fatty animals with preconditioning (3417 U/L) had drastic reduced AST levels, while the AST levels remained high in fatty animals without preconditioning (13466 U/L). Apoptosis was the predominant form of cell death in the lean liver with 77% Tunel pos. hepatocytes at 4 h reperfusion compared with only 17% in fatty livers and 5% in fatty livers with ischemic preconditioning (p < 0.001 each). Caspase 3 activity, as a key mediator of apoptosis, was 3-fold higher in lean animals at 4 h reperfusion when compared to the fatty group without ischemic preconditioning (p < 0.01 each). 24 h after reperfusion 85% of the liver tissue was necrotic in fatty animals, compared with 18% necrosis in the lean control (p< 0.01). Ischemic preconditioning of fatty mice reduced the amount of necrosis to 30% (p < 0.01). Hepatic steatosis was associated with a significant decrease of tissue ATP levels after 4 h and 24 h reperfusion when compared with the lean group (1.29 vs. 0.76 & 1.06 vs. 0.39 μmol/pg; p = 0.04 & p = 0.007). Ischemic preconditioning of fatty livers increased the tissue ATP levels significantly at both time points (1.06 & 0.83 μmol/pg;p < 0.05 each). Conclusion: Ischemic preconditioning effectively protects the steatotic liver by reducing apoptotic and necrotic liver injury. Ischemic reperfusion in steatotic livers is associated with low ATP levels, which can be improved by ischemic preconditioning.

Kalte Ischämie senkt die Regenerationsfähigkeit der Leber: Neue Aspekte für Split-Leber und Lebendspenden Transplantation

Die Lebertransplantation ist die einzige therapeutische Option fur Patienten mit fortgeschrittenen Lebererkrankungen. Kurzlich wurden partielle Lebertransplantationen, wie z. B. Split-Leber oder Lebendspenden Transplantation zur Verminderung des dramatischen Organmangels entwickelt. Der Effekt von kalter Ischamie, wie sie bei der Organkonservierung auftritt, auf die Regenerationsfahigkeit der Leber ist unbekannt. Wir stellten die Hypothese auf, dass kalte Ischnerationamie die Regenerationsfahigkeit von Hepatozyten reduziert und zu einer erhohten Mortalitat nach Lebertransplantation fuhrt. Methoden: Lewis Ratten wurde einer 70% Leberteilresektion unterzogen (Ko) und mit Geschwistertieren verglichen, die einer totalen Hepatektomie und einer partiellen Lebertransplantation (30%) unterzogen wurden. Vor der Transplantation wurde das Lebergewebe 30 Minuten, 10 Stunden oder 16 Stunden bei 4°C in UW-Losung konserviert. 2 und 4 tage nach resektion bzw partieller Transplantation wurde die Leberregeneration (PCNA-Farbung: Gl-Phase, BrdU-Farbung: S-Phase, Mitosen: M-Phase jeweils pro high power Feld) bestimmt. Resultate: 70% Leberresektion war mit 100% uberleben der Tiere assoziiert, wahrend nach 30 Minuten, 10 Stunden und 16 Stunden Konservierung und Transplantation 75%, 61% und 26% der Versuchstiere dauerhaft uberlebten. Kurzzeitige kalte Ischamie (30 Min) und Transplantation fuhrte zu einer signifikanten Verbesserung der Regeneration nach 2 Tagen im Vergleich zu Kontrolltieren nach 70% Resektion (PCNA 140 vs 80; BrdU 42 vs 27, Mitosen 5 vs 3,5 p < 0.01 jeweils). Nach 4 Tagen war die Regeneration in Tieren nach 70% Resektion und 30 Min. kalter Ischamie und Transplantation identisch. 10 und 16 Stunden kalte Ischamie vor Transplantation fuhrte zu einer dramatischen Reduktion aller 3 Parameter der Leberregeneration nach 2 Tagen (PCNA 53 & 38; BrdU 12 & 5; Mitosen 1.5 und 0,5) und 4 Tagen (PCNA 398c 15; BrdU 21 & 5; Mitosen 0.5 & 0,2) im Vergleich zu der Kontrollgruppe mit alleiniger Resektion und zu Tieren mit 30 Min. Konservierung. Die Dauer der Konservierung korrelierte dabei mit dem Ausmass der Regenerationsblockade. Schlusfolgerung: Kurzzeitige kalte Ischamie verbbessert die Regenerationsfahigkeit der Leber. Im Gegensatz dazu bewirkt eine langere kalte Ischamie eine deutliche Reduktion der hepatozellularen Regeneration. Die Vermiderung der Regenerationsfahigkeit nach kalter Ischamie korreliert mit einer hohen Mortalitat nach partieller Transplantation verbunden. Verlangerte kalte Ischamizeiten sollten bei partieller Lebertransplantation vermieden werden.