Gastroenterology
Volume 133, Issue 2 , Pages 713-716, August 2007

Seek and Hide Phosphatidylserine: A New Approach to Prevent Hepatic Ischemia/Reperfusion Injury

  • Emanuele Albano

      Affiliations

    • Corresponding Author InformationAddress requests for reprints to: Emanuele Albano, MD, Department of Medical Sciences, University “Amedeo Avogadro” of East Piedmont, via Solaroli 17, 28100 Novara, Italy.

Department of Medical Sciences, University “Amedeo Avogadro” of East Piedmont, Novara, Italy

Article Outline

 

See “Diannexin, a novel Annexin V homodimer, provides prolonged protection against hepatic ischemia-reperfusion injury in mice,” by Teoh NC, Ito Y, Field J, et al, on page 632.

Ischemia/reperfusion injury is the primary cause for hepatic failure after hemorrhagic shock, temporary clamping of the hepatoduodenal ligament during liver resection (Pringle maneuver), and liver transplantation.1 Despite improvements in procedures for liver preservation, hepatic ischemia/reperfusion remains an important clinical problem. Furthermore, because of the shortage of organs, steatotic livers that are more susceptible to re-oxygenation damage and partial livers obtained from living donors or split grafts are increasingly used for transplantation, thus increasing the risks related to graft reperfusion injury.2, 3 The pathogenesis of hepatic ischemia/reperfusion injury is complex and several factors contribute to tissue damage. The lack of oxygen during the ischemic period causes the depletion of cellular ATP, mitochondrial de-energization, and alterations of H+, Na+, and Ca2+ homeostasis that activate hydrolytic enzymes and impair cell volume regulation.4, 5 Upon re-oxygenation, the formation of reactive oxygen species (ROS) by uncoupled mitochondria promotes oxidative stress and mitochondrial permeability transition.4, 6 The combination of these events is responsible for cell death by either necrosis or apoptosis.4, 6 Concomitantly, the activation of Kupffer cells releases ROS, nitric oxide (NO), eicosanoids, and pro-inflammatory cytokines/chemokines (tumor necrosis factor [TNF]-α, interleukin [IL]-6, IL-1β, MCP-1, IL-12, and IL-8).7 These pro-inflammatory mediators, in concert with the increased expression of adhesion molecules (ICAM-1, VCAM-1, and P- and E-selectins) by sinusoidal endothelial cells (SECs), promote liver infiltration by granulocytes and monocytes that, upon activation, further contribute to the progression of parenchymal injury.7, 8 The hepatic production of pro-inflammatory cytokines/chemokines, particularly TNF-α, also propagates the inflammatory response to neighboring organs such as the lung, causing pulmonary insufficiency.7

In recent years, increasing efforts have been directed to mitigate or prevent hepatic ischemia/reperfusion injury.8, 9, 10 The strategies proposed have focused on (1) improving the procedures of graft harvesting and cold preservation; (2) enhancing the tolerance to ischemia/reperfusion damage through the induction of liver preconditioning; and (3) interfering with the events that are associated with the development of irreversible cell injury. Among these strategies, the induction of preconditioning by either exposing the liver to brief periods of ischemia followed by a transient reperfusion or the direct administration of mediators, such as adenosine, ATP, NO, atrial natriuretic peptide, and cardiotrophin, capable of triggering the preconditioning responses, has been effective in many experimental conditions.8, 11, 12, 13 Moreover, several clinical studies demonstrate the benefits of hepatic ischemic preconditioning in the setting of liver resection and transplantation.14 The main advantage of preconditioning is the induction of physiologic responses that increase the tolerance of both parenchymal and nonparenchymal cells to ATP depletion, oxidative stress, and pro-apoptotic signals.11 Moreover, preconditioning down-modulates inflammatory reactions and preserves hepatic microcirculation,11 which may be effective in steatotic livers,15 and enhances hepatic regeneration.16 These latter observations suggest a possible application of preconditioning in alleviating the risk of the “small-for-size syndrome” after liver transplant from living donors of split grafts. On the other hand, the application of preconditioning procedures during graft harvesting does not guarantee that optimal protection could be maintained until the graft reperfusion.

The pharmacologic modulation of the events responsible for causing hepatic reperfusion damage has been pursued by using agents that reduce and/or antagonize oxidative stress, mitochondrial permeability transition, pro-apoptotic signals, inflammatory mediators, and microcirculatory disturbances (Figure 1)8, 9, 10, 11, 17 (refer also to www.ingentaconnect.com18). Although most of these agents are effective in reducing experimental hepatic reperfusion injury; their possible application in clinical settings has often been rather disappointing.9, 10

  • View full-size image.
  • Figure 1. 

    Events responsible for the pathogenesis of hepatic ischemia/reperfusion injury and mechanisms of action of some pharmacologic agents that have been reported to confer protection against liver damage.8, 9, 10 Hypoxia causes the depletion of cellular ATP, mitochondrial de-energization, alterations of H+, Na+, and Ca2+ homeostasis and conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XOD). Upon re-oxygenation, the formation of ROS by uncoupled mitochondria and XOD promotes oxidative stress and mitochondrial permeability transition that leads to cell death by either necrosis or apoptosis. Concomitantly, the activation of Kupffer cells releases ROS, NO, eicosanoids and pro-inflammatory cytokines/chemokines (TNF-α, IL-6, IL-1β, MCP-1, IL-12, IL-8). These pro-inflammatory mediators, in concert with the increased expression of adhesion molecules (ICAM-1, VCAM-1 P- and E-selectins) by sinusoidal endothelial cells, promote liver infiltration by granulocytes and monocytes. CD4+ T lymphocytes can also contribute to hepatic reperfusion injury.

In this issue of Gastroenterology, Teoh et al19 report on a new and original approach to the prevention of liver ischemia reperfusion by using Diannexin, a homodimer of the human recombinant annexin A5 that was produced using molecular biology techniques.19 Diannexin has the advantage over the native protein of having a higher molecular weight that reduces its otherwise rapid renal clearance.20 The action of Diannexin on warm hepatic ischemia/reperfusion injury was investigated in female C57BL6 mice using a well-established in vivo experimental protocol consisting in a 90-minute interruption of the perfusion to the left lateral and median liver lobes followed by 2 or 24 hours of reperfusion. The administration of Diannexin (100–1000 μg/kg) 5 minutes or 24 hours before or 1 hour after ischemia/reperfusion almost completely prevented ALT leakage during the early phase of reperfusion injury (2 hours) and protected from the typical reperfusion-induced liver necroinflammation. The protective effects of Diannexin were associated with a reduction in the swelling and the detachment of SECs. Diannexin also decreased hepatic mRNA expression of pro-inflammatory molecules (MIP-2, ICAM-1, VCAM) and almost abolished leukocyte and platelet adherence to SECs.19

Annexin A5 is a member of the annexin family that include >160 proteins sharing the property of binding in a Ca2+-dependent manner to negatively charged phospholipids, namely phosphatidylserine, phosphatidylcholine, and phosphatidic acid.21 Because of this property, annexin A5 is widely used as a marker for apoptotic cells.22 Moreover, annexin A5 present in the blood and extracellular fluids contributes to the regulation of hemostasis, particularly in the placental circulation, where it interacts with phosphatidylserine-expressing syncytiotrophoblast cells.23 Diannexin has an higher affinity for phosphatidylserine than annexin 5A and shares the anticoagulant, antithrombotic, and apoptotic cell-binding properties of the native homolog.20

Although Teoh et al do not provide detailed insight into the mechanisms responsible for Diannexin protection against reperfusion injury, the evidence presented suggests that Diannexin mainly acts by ameliorating microcirculatory disturbances caused by leukocyte and platelet adherence to damaged SECs.19 The exposure of phosphatidylserine on the outer layer of the cell plasma membrane is a characteristic feature of apoptosis and is evident even before the definitive commitment to death.24 SEC apoptosis is an early feature of reperfusion injury following both cold and warm liver ischemia.25, 26 Several studies in experimental animals and in humans have shown that liver ischemia/reperfusion promotes the activation of complement that, in turn, contributes to liver damage by recruiting and activating phagocytes.27 Interference with complement activation using selective inhibitors has been shown to prevent tissue injury after ischemia/reperfusion.28 Phosphatidylserine exposed on the surface of apoptotic endothelial cells is an important trigger of the complement cascade.29 Thus, by “seeking then hiding” phosphatidylserine on SEC plasma membranes, Diannexin can prevent complement-mediated signals responsible for the progression of inflammation in reperfused livers.7, 27, 28

In recent years, growing evidence points to the importance of cell-derived microparticles in promoting inflammatory and thrombotic responses following vascular dysfunction.30, 31 Microparticles are small (0.5–1.0 μm) cell-derived vesicles shed from the blebbing of the plasma membranes of platelets, lymphocytes, endothelial cells, and smooth muscle cells when they are activated by agonists, shear stress, or undergo apoptosis.30 Microparticles harbor cell plasma membrane proteins and exhibit at their surface negatively charged phospholipids, chiefly phosphatidylserine, which accounts for their procoagulant and pro-inflammatory properties.30 Elevated levels of circulating microparticles have been detected in acute coronary ischemia, myocardial infarction, diabetic vasculopathies, eclampsia, and severe trauma, and are thought to contribute to leukocyte and platelet adhesion, thrombosis, and vascular dysfunctions associated with these diseases.31 The involvement of microparticles in the pathogenesis of hepatic reperfusion injury has not been investigated so far. However, extensive blebbing of hepatocyte and SEC plasma membranes is an early feature of cellular distress during hypoxia/reperfusion.3 Thus, it is possible that microparticles originating in these conditions might contribute to the cause of inflammatory responses within reperfused livers. Because annexin A5 efficiently binds to microparticles,31 it is feasible that the protective action of Diannexin on hepatic reperfusion injury might also involve the scavenging of microparticles originating from the shedding of various liver resident injured cells, thereby preventing the vascular and inflammatory responses that worsen parenchymal damage after liver reperfusion.7 In this context, the observations by Teoh et al19 provide a well-appreciated and timely stimulus to further investigate the role that the SEC responses to hypoxia/reoxygenation stress play in the pathogenesis of hepatic reperfusion damage.

While we await further confirmation of the capacity of Diannexin to ameliorate reperfusion injury in transplanted livers, the work by Teoh et al gives new hope that in the near future the combined application of specific protective agents and improvements in preconditioning procedures will minimize hepatic reperfusion damage, thus safely increasing the use of marginal or split livers for transplantation.

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PII: S0016-5085(07)01171-7

doi:10.1053/j.gastro.2007.06.030

Refers to article:

  • Diannexin, a Novel Annexin V Homodimer, Provides Prolonged Protection Against Hepatic Ischemia-Reperfusion Injury in Mice , 22 May 2007

    Narci C. Teoh, Yoshiya Ito, Jacqueline Field, Nancy W. Bethea, Deama Amr, Margaret K. McCuskey, Robert S. McCuskey, Geoffrey C. Farrell, Anthony C. Allison
    Gastroenterology August 2007 (Vol. 133, Issue 2, Pages 632-646)

Gastroenterology
Volume 133, Issue 2 , Pages 713-716, August 2007

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