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

GASTROENTEROLOGY 2007;133:632– 646

Diannexin, a Novel Annexin V Homodimer, Provides Prolonged Protection Against Hepatic Ischemia-Reperfusion Injury in Mice NARCI C. TEOH,* YOSHIYA ITO,‡ JACQUELINE FIELD,* NANCY W. BETHEA,‡ DEAMA AMR,* MARGARET K. MCCUSKEY,‡ ROBERT S. MCCUSKEY,‡ GEOFFREY C. FARRELL,* and ANTHONY C. ALLISON§ *Storr Liver Unit, Westmead Millennium Institute, University of Sydney at Westmead Hospital, Westmead, NSW, Australia; ‡Department of Cell Biology and Anatomy, University of Arizona, Tucson, Arizona; and §Alavita Inc., Mountain View, California

See editorial on page 713.

BASIC–LIVER, PANCREAS, AND BILIARY TRACT

Background & Aims: Ischemia-reperfusion injury (IRI) remains an important cause of liver failure after hepatic surgery or transplantation. The mechanism seems to originate within the hepatic sinusoid, with damage to endothelial cells, an early, reproducible finding. Sinusoidal endothelial cells (SECs), damaged during reperfusion, activate and recruit inflammatory cells and platelets. We hypothesized that a recombinant human annexin V homodimer, Diannexin, would protect SECs from reperfusion injury. Methods: We tested this proposal in a well-characterized in vivo murine partial hepatic IRI model. Results: Whether administered 5 minutes or 24 hours before or 1 hour after ischemia-reperfusion, Diannexin (100 –1000 ␮g/kg) almost completely protected against liver injury. The protective efficacy conferred by Diannexin was highly visible at the microcirculatory level. Thus, although IR in this model is associated with early swelling and gap formation in SECs, Diannexin ameliorated these effects as shown by >80% reduction in alanine aminotransferase values during the early phase of reperfusion injury (2 hours) and near normalization of liver necrosis and inflammation in the late phase of inflammatory recruitment (24 hours). Consistent with the proposed role of SEC injury in hepatic IRI, Diannexin also decreased hepatic expression of proinflammatory molecules (MIP-2, ICAM-1, VCAM), abolished leukocyte and platelet adherence to damaged SECs, and, by in vivo microscopy, Diannexin preserved microcirculatory blood flow and hepatocyte integrity during reperfusion. Conclusions: Diannexin is an apparently safe therapeutic protein that provides prolonged protection against hepatic IRI via cytoprotection of SECs, thereby interrupting secondary microcirculatory inflammation and coagulation.

I

schemia-reperfusion injury (IRI) occurs when the blood supply to an organ is cut off and later restored. In the liver, IRI remains a major factor contributing to organ failure after hepatic surgery for liver

cancer and is a challenging problem for other types of hepatobiliary surgery.1 IRI may also contribute to the mechanism of liver injury with shock, cardiac failure, some drugs, and toxins.2 A similar form of injury, cold preservation injury, contributes importantly to acute graft failure that complicates 5%–10% of liver transplantation operations.3 Although research into pathogenic mechanisms for IRI has identified multiple candidate injury and inflammatory pathways, there is not yet any generally applicable therapeutic strategy to prevent or treat this disorder.3,4 Development of a biosynthesized homodimer of human annexin V, Diannexin was predicated on a conceptual basis for the pathogenesis of IRI that involves a network of proinflammatory and procoagulative events within the microcirculation.4 In culture, sinusoidal endothelial cells (SECs) are the only liver cell type to undergo posthypoxic reoxygenation injury,5 and, in the intact liver, SEC injury is a prominent manifestation of both warm reperfusion injury or cold preservation injury.6 – 8 The physiologic asymmetry of the plasma membrane phospholipid bilayer, with phosphatidylserine (PS) confined to the cytoplasmic layer,9 is maintained through an adenosine triphosphate (ATP)-requiring phospholipid translocase.10 It seems likely that ATP depletion resulting from tissue ischemia would allow some PS to translocate to the outer (vascular) layer of the plasma membrane, at which it is known to play a role in activation and recruitment of both platelets and inflammatory cells. Annexin V binds to such everted PS residues and is an early marker of regulated cell death at a time when cell injury may be reversible.11 Culturing other types of endothelial cells under hypoxic conditions increases annexin V binding,12 and the process of PS translocation to the surface of Abbreviations used in this paper: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICAM-1, intercellular adhesion molecule; IRI, ischemia reperfusion injury; KC, Kupffer cell; MIP-2, macrophage inflammatory protein-2; PS, phosphatidylserine; SEC, sinusoidal endothelial cell; SEM, scanning electron microscopy; sPLA2, phospholipase A2; TEM, transmission electron microscopy; TNF, tumor necrosis factor; TxB2, thromboxane B2; VCAM, vascular cell adhesion molecule. © 2007 by the AGA Institute 0016-5085/07/$32.00 doi:10.1053/j.gastro.2007.05.027

endothelial cells is augmented by tumor necrosis factor-␣ (TNF-␣) and other proinflammatory cytokines, which increase during the onset of hepatic IRI.13–16 Our proposal is that ischemia results in changes to the hepatic SEC so that during reperfusion, bloodborne cells are activated and bind to the damaged surface of this cell type to trigger downstream proinflammatory and procoagulant effects, particularly through the recruitment of other cell types, such as leukocytes and platelets. Such recruitment is known to reduce blood flow in the hepatic microcirculation and exacerbate ischemic injury to the hepatocytes. Because annexin V has a short serum half-life (t1/2) (less than 20 minutes), thereby limiting the practical application of any therapeutic efficacy, we constructed a synthetic annexin V homodimer, Diannexin, which has a prolonged serum t1/2 compared with native annexin V. In the present study, we tested whether Diannexin protects against hepatic IRI. Having shown impressive protection against liver injury and hepatic inflammation over a 2-hour to 24-hour time course of reperfusion, we then examined the effects of Diannexin on the hepatic microcirculation. We found that Diannexin protects hepatic SECs from swelling and gap formation, blocks multiple proinflammatory pathways, and abrogates both inflammatory cell recruitment and platelet aggregation. The functional outcome of these pharmacodynamic effects is to preserve hepatic microcirculatory blood flow, which is usually greatly impaired by the inflammatory changes within the hepatic sinusoid during IRI.

Materials and Methods Preparation of Diannexin The sequence of amino acids in annexin V and Diannexin are shown in Supplemental Figure 1 (See Supplemental Figure 1 online at www.gastrojournal. org). The N-terminus of one annexin V sequence was joined by a 14 amino acid linker to the C-terminus of a second annexin V sequence. The composition of the linker was designed to meet 4 requirements. First, from the 3-dimensional structure of annexin V, and Ca2⫹ and PS-binding sites determined by x-ray crystallography and site-specific mutagenesis, we estimated the length of linker required for the dimer to fold in such a way that all Ca2⫹ and PS-binding sites could come into contact with PS on a cell or microvesicle surface. Second, the linker has no ␣-helical or other secondary structure that limits flexibility. Third, a glycine-serine swivel near each end allows some rotation around the plane of the linker. Fourth, the amino acids in the linker form peptides of low immunogenicity. The resultant molecular weight of Diannexin is 73,137 daltons. Diannexin was produced by ATG Laboratories (Eden Prairie, MN) where the protein was expressed in

DIANNEXIN AND HEPATOPROTECTION

633

Figure 1. Experimental protocols for hepatic IRI and administration of Diannexin. All animals were subjected to 90-minute ischemia followed by reperfusion for the indicated times (2 hours, 24 hours). Diannexin was injected intravenously either 5 minutes, 6 hours, 24 hours, or 48 hours prior to IR (arrow in A) or 10 to 60 minutes after onset of reperfusion (arrow in B). Blood and liver samples were collected at 2-hour or 24-hour reperfusion. Drawings are not to scale.

Escherichia coli BL21 cells, then purified from aqueous soluble extracts by anion exchange chromatography using a UNO Sphere Q Column (Bio-Rad, Hercules, CA), followed by heparin affinity chromatography using Hi Trap Heparin HP columns from Amersham Biosciences (Piscataway, NJ). SDS polyacrylamide gel electrophoresis and Coomassie brilliant blue staining showed a single peak at 73 kilodaltons. Matrix assisted laser desorption ionization-time of flight (MALDITOF) mass spectrometry was accomplished using a PerSeptive Biosystems Voyager-DE Pro workstation (Foster City, CA) operating in linear positive ion mode with a static accelerating voltage of 25 kV and a delay time of 40 ns. MALDI-TOF mass spectrometry showed a single peak at 73 kilodaltons, with second and third peaks (⫹2 and ⫹3) at 36.6 kilodaltons and 24.3 kilodaltons.

Murine Model of Partial Hepatic IRI and Administration of Diannexin All animal protocols and studies complied with the highest International Criteria of Animal Experiments and were approved by the Western Sydney Area Health Service (WSAHS) Animal Ethics Committee. Female C57BL6 wild-type mice weighing 18 to 25 g were fed a commercial pellet diet and allowed water ad libitum. The design of the IRI experiments is shown in Figure 1. Mice were anesthetized (ketamine 100 mg/kg and xylazine 20 mg/kg body weight, administered intraperitoneally) and subjected to occlusion of the left lateral and median lobes of the liver by applying an atraumatic microvascular clamp to the vascular pedicle for 90 minutes.17,18 Reperfusion was established by removal of the vascular clamp, and animals were allowed to recover. After 2 or 24 hours of reperfusion, animals were killed by exsanguination, blood was col-

BASIC–LIVER, PANCREAS, AND BILIARY TRACT

August 2007

634

TEOH ET AL

GASTROENTEROLOGY Vol. 133, No. 2

Table 1. Summary of Diannexin Plasma Toxicokinetic Parameters Dosage (mg/kg)

Sex

Cmaxa (ng/mL)

tmax(h)

tlast(h)

AUC (ng/h/mL)

0.5 0.5 1.0 1.0

Male Female Male Female

8720 9650 100,000 44,700

0 0 0 0

8 8 8 8

3750 3840 21,900 11,900

AUC, area under the curve; Cmax, maximum plasma concentration; tmax, time to maximum concentration; and tlast, time of the last measured concentration. aConcentrations at time 0 were estimated by extrapolation of observed data.

BASIC–LIVER, PANCREAS, AND BILIARY TRACT

lected via cardiac puncture for preparation of plasma, and livers were harvested for tissue studies. One group of control mice was subjected to anesthesia and sham laparotomy. Liver samples were collected from the medial (postischemic) lobes and immediately frozen in liquid nitrogen or fixed in phosphate-buffered formalin prior to embedment in paraffin. In some experiments, the “control” protein, annexin V monomer (1000 ␮g/kg per body weight) or Diannexin was administered via lateral tail vein injection to mice at varying doses of 10 –1000 ␮g/kg per body weight 5 minutes prior to partial hepatic IRI (90-minute ischemia and reperfusion for 2 or 24 hours) (n ⫽ 5 up to 8 mice at each time point per treatment group). In other experiments, the duration of protection was assessed: Diannexin (1 mg/kg) was injected intravenously (either 6 or 24 hours, as indicated by arrow in Figure 1A) before clamping the hepatic pedicle vasculature, or 10 to 60 minutes after reperfusion (arrow in Figure 1B). Saline vehicle controls were studied for each experimental time point (n ⫽ 4).

microscopy using a charge-coupled device (CCD) camera (MTI, Michigan, IN), video monitor, and Sony Betacam video tape recorder (Sony Medical Electronics, Park Ridge, NJ). The liver was exteriorized through a left subcostal incision and positioned over a window of optical-grade mica in a specially designed tray mounted on a microscopic stage. The tray provided for the drainage of irrigating fluids, and the window overlaid a long working distance condenser. The liver was covered by a piece of Saran wrap (Dow Chemical Co., Midland, MI), which held it in position and limited movement. Homeostasis was ensured by a constant irrigation of the liver with Ringer’s solution maintained at body temperature. With the X80/NA 1.00 water immersion objective (Leitz, Wetzlar, Germany) employed for these studies, the resolution was 0.3 to 0.5 ␮m. Microvascular events were observed and recorded in naïve or Diannexin-treated mice subjected to 90-minute ischemia and 30- to 120-minute reperfusion (n ⫽ 5 per experimental group). The phagocytic function of hepatic macrophages was assessed by measuring the uptake by individual cells of fluorescent 1.0-␮m latex particles (Fluoresbrite-fluorescent monodispersed polystyrine microspheres; Polysciences, Warrington, PA: excitation 490 nm/emission 530 nm) by individual cells. The latex particles were diluted 1:10 with sterile saline and injected into a mesenteric vein using a 30-gauge lymphangiography needle (Becton Dickinson, Franklin Lakes, NJ). The distribution and relative numbers of macrophages was measured by counting the number of cells that phagocytosed latex particles in a standardized microscopic field (4125 ␮m2) 15 minutes after injection. To assess regional distribution, the number of phagocytic Kupffer cells per microscopic field was counted in 10 periportal and 10 centrilobular regions in each animal. The relative adequacy of

Assessing the Severity of Liver Injury Severity of liver injury was determined from serum levels of alanine aminotransferase (ALT) and liver histology. Serum ALT was assayed by the Department of Clinical Chemistry, Institute for Clinical Pathology and Medical Research, WSAHS, using automated procedures. Consecutive liver sections (4 ␮m thick) were cut from paraffin-embedded liver and stained with H&E for evaluation of the extent of hepatic necrosis. Areas of hepatic necrosis were measured in 5 low-power fields (⫻4 magnification) using the Optimas 6.5 Image Analysis software (Media Cybernetics, Silver Spring, MD). The extent of leukocyte infiltration in liver sections was appraised qualitatively.

In Vivo Microscopy Hepatic microvascular alterations were examined using established high-resolution in vivo microscopic methods under ketamine/xylazine anesthesia, as reported previously.19 A compound binocular microscope adapted for in vivo microscopy was equipped to provide either transillumination or epiillumination, as well as video

Figure 2. Blood radioactivity disappearance curve after intravenous injection of Diannexin, with the ␣- and ␤-phases superimposed following injection of labelled Diannexin in rats.

DIANNEXIN AND HEPATOPROTECTION

635

Figure 3. Diannexin is hepatoprotective against IRI. (A) Hepatic IRI in naïve and Diannexin pretreated mice. Animals were administered Diannexin 1 mg/kg body weight intravenously (IV) at indicated times prior to IR. S, sham-operated mice. Serum ALT levels were determined after 24 hours of reperfusion. Graphs denote mean values; error bars are ⫾ SD for 5 mice in each group. *P ⬍ .001 compared with naïve/saline-treated controls. (B) Hepatoprotective effect of low-dose Diannexin. Diannexin was administered at varying doses IV 6 hours before ischemia. Serum ALT levels were determined at 24-hour reperfusion. Results are mean ⫾ SD, n ⫽ 4 animals per experimental group. *P ⬍ .01 compared with saline-treated controls. (C) Diannexin provides hepatoprotection when administered during 10 minutes (D10minR) or up to 60 minutes (D60minR) of reperfusion. Serum ALT levels were determined at 24-hour reperfusion. Results are mean ⫾ SD, n ⫽ 4 animals per experimental group. *P ⬍ .01 compared with saline-treated controls (naïve). (D) Liver histology from Diannexin pretreated and (E) naïve mice subjected to 90-minute hepatic ischemia and 24-hour reperfusion. H&E staining of representative liver sections is shown at 200⫻ magnification. Necrotic areas (N) are indicated. Similar results were obtained with Diannexin (1 mg/kg body weight) administered 24 hours before IR. Sections are representative of n ⫽ 4 experiments.

BASIC–LIVER, PANCREAS, AND BILIARY TRACT

August 2007

636

TEOH ET AL

GASTROENTEROLOGY Vol. 133, No. 2

Table 2. Hepatic Ischemia Reperfusion (IR) Injury, Expressed as Area of Hepatic Necrosis, at 24-Hour Reperfusion Following 90-Minute Ischemia Experimental group Naïve Diannexin 5 minutes prior to IRa Diannexin 6 hours prior to IR Diannexin 24 hours prior to IR

% Area of necrosis 20 ⫾ 3.7 0b 0b 0.8 ⫾ 0.3b

NOTE. Results are mean ⫾ SD of 5 mice in each group; area of necrosis expressed as a percentage of total area of liver section at 40⫻ magnification. IR, ischemia reperfusion. aWhen injected into sham-operated mice (that is, in the absence of IR), Diannexin at 1 mg/kg body weight did not increase ALT at 24 hours or cause development of hepatic necrosis. bP ⬍ .0001 compared with naïve animals subjected to IR.

BASIC–LIVER, PANCREAS, AND BILIARY TRACT

blood perfusion through the sinusoids was evaluated by counting the number of sinusoids exhibiting blood flow in the same microscopic fields in which the numbers of phagocytic macrophages were determined. Transmitted bright-field and epiilluminated fluorescence images were obtained simultaneously to permit imaging of fluorescent latex bead phagocytosis and provide images of the sinusoidal wall and blood flow simultaneously. Because reduced perfusion of individual sinusoids can limit the delivery of the latex particles to phagocytic cells within those vessels, the ratio of macrophages that phagocytosed latex particles to sinusoids in which blood flowed was used as an overall mean index of phagocytic activity of macrophages per microscopic field. To examine the interaction of leukocytes with the sinusoidal wall, quantification of leukocytes adhering to the endothelial lining of sinusoids was calculated by counting the number of leukocytes per 100-␮m length of sinusoid in the same microscopic fields. A leukocyte was defined as adherent to the sinusoidal wall if it remained stationary for at least 30 seconds. We also counted the number of accumulated platelets in the sinusoids per microscopic field. A platelet was defined as adherent if it remained stationary for more than 30 seconds. Concomitantly, platelets were labelled by intravenous injection of Rhodamine 6G (0.1 mL, 10 mg/mL, Sigma) and visualized by epiillumination with filter combination of 510 to 560 nm. The use of transillumination and epiillumination in the same microscopic unit is helpful to detect platelets. The swelling of SECs, which is an indication of activation and/or injury, was assessed by counting the numbers of swollen cells whose nuclear regions protruded across one third or more of the lumen in the same microscopic fields. The results were averaged, and the data were represented as the average numbers of the parameters in each animal.

Electron Microscopy In a separate set of experimental animals, routine methods were used to prepare liver specimens for scan-

ning (SEM) and transmission electron microscopy (TEM).19 Livers were fixed by perfusion of portal veins with 0.1 mol/L cacodylate buffer to wash out blood and were subsequently fixed with 1.5% glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.4. For TEM, small pieces of liver were washed in buffer, postfixed with 1% osmium tetroxide in 0.1 mol/L cacodylate buffer at 4°C, dehydrated through a graded series of ethanol solutions, briefly rinsed in propylene oxide, and embedded in epoxy resin. Thin sections were cut on a Reichert Ultracut microtome (Leica, Deerfield, IL) and examined and photographed using a Philips CM-12S electron microscope (Philips Electronic Instruments, Mahwah, NJ). For SEM, pieces of perfused-fixed livers were dehydrated in a graded ethanol series, critical point dried, fractured, sputter coated with 10 nm gold, and examined using an FEI XL35 SEM.

Measurement of Hepatic Intercellular Adhesion Molecule-1, Vascular Cell Adhesion Molecule, and Macrophage Inflammatory Protein-2 Messenger RNA by Real-Time Polymerase Chain Reaction First-strand complementary DNA was prepared by reverse transcription of 5 ␮g total liver RNA using 100 U SuperScript II RNaseH⫺ reverse transcriptase (Life Technologies) and 0.5 ␮g random hexamers (Promega, Madison, WI). Polymerase chain reactions (PCRs) for intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM), macrophage inflammatory protein (MIP)-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplification contained 10 ng reverse-transcribed total RNA, primers, and Taqman probes in Universal PCR master mix (PE Applied Biosystems, Foster City, CA). The ABI Prism 7700 Sequence Detection System (Otago, New Zealand) was used for real-time detection of the amplification product. The amount of mRNA was calculated by reference to a calibration curve and expressed as the ratio of ICAM-1, VCAM, or MIP-2 to GAPDH (regarded as the invariant control).

Western Blot Analysis of ICAM-1 Liver homogenates in protein lysis buffer (20 mmol/L Tris, 0.5 mmol/L MgCl2, 1 mmol/L DTT, 0.02% NaN3, and a mixture of protease and phosphatase inhibitors) were resolved by 12% SDS-PAGE under reducing conditions. The conditions for detecting ICAM-1 (Santa Cruz Biotechnology Inc, Santa Cruz, CA) protein by immunoblotting have been reported.20

ICAM-1 Immunohistochemistry Paraffin-embedded liver sections were quenched for endogenous peroxidase activity using 3% hydrogen peroxide, after which antigen retrieval was performed with heated citric acid buffer. Slides were incubated with 1:100 dilution of hamster ICAM-1 (BD Sciences Pharmingen, Palo Alto, CA) for 1 hour at room temperature.

Detection of the primary antibody was carried out using the appropriate biotinylated antibody (Vector Laboratories, CA) and peroxidase DAB kit (Ventana, Tucson, AZ).

Measurement of Hepatic Thromboxane B2 Levels Thromboxane B2 (TxB2) levels in whole liver homogenates were determined by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Assay Designs, Ann Arbor, MI).

Statistical Analyses The Student t test and 2-way analysis of variance (ANOVA) were used for the comparison of data from different treatment groups. The results are presented as mean ⫾ SD and are considered significant when P is less than .05.

Results Development of Diannexin as a Therapeutic Protein and Concentration of Diannexin In Vivo Several serine proteases of blood coagulation cascades (factors X, Xa, and Va) use externalized PS as a docking site.21–23 Pretreatment with annexin V, a human placental anticoagulant competes for PS binding so as to prevent the activation of these enzymes, thus exerting anticoagulant activity without increasing hemorrhage.22–26 We used recombinant DNA technology to express in Escherichia coli 2 human annexin V sequences joined by a 14 amino acid flexible linker (see Materials and Methods and Supplemental Figure 1 online at www. gastrojournal.org) to obtain a protein large enough to exceed the renal filtration threshold, thereby extending its survival in the circulation.27 Because the mouse circulatory volume is too small for serial sampling, survival of Diannexin in the circulation was monitored in Sprague-Dawley rats as part of a toxicokinetic study. Following a single intravenous injection of Diannexin, blood samples were obtained by tail vein bleeds, and plasma concentrations of Diannexin were determined by a 2-site immunoassay. The findings are summarized in Table 1. Furthermore, 125 I-labelled Diannexin when injected into rats had a t1/2 (␤ phase) of 6.5 hours (Figure 2). These data confirm that Diannexin has a longer half-life in the circulation than monomeric annexin V. The dimer (Diannexin) also has a higher affinity for PS on cell surfaces than does physiologic monomeric annexin V28 (see Supplemental Figure 2 online at www. gastrojournal.org). Because externalized PS is a docking site for secreted isoforms of phospholipase A2 (sPLA2; which are elaborated in inflammatory states),29 –31 we performed studies to confirm that Diannexin (in the dose range used in IRI experiments, 100 to 1000 ␮g/kg, IV) was a potent inhibitor of prothrombinase and sPLA2

DIANNEXIN AND HEPATOPROTECTION

637

activities and that the dimer inhibited venous thrombosis in rats28 (see Supplemental Figures 3–5 online at www. gastrojournal.org).

Diannexin Protects Against Hepatic IschemiaReperfusion Injury in Mice To test the efficacy of Diannexin against hepatic IRI, we used a well-characterized in vivo partial hepatic ischemia-reperfusion model in female mice (which have lower mortality than male mice subjected to IRI).13–15,17,18 Figure 1 outlines the experimental protocols employed. Following 24 hours of postischemia reperfusion, there is substantial liver damage in this model as indicated by significantly (⬃20-fold) elevated serum ALT levels (Figure 3A) and necrotic areas surrounded by inflammatory cells quantified in histologic sections (Table 2). Diannexin (1 mg/kg body weight) was injected intravenously at 5 minutes or earlier times before vascular clamping in this model and proved highly protective against IRI, as shown by ALT levels (Figure 3A). The hepatoprotective efficacy of Diannexin was retained at 2-hour reperfusion (see Supplemental Figure 6A online at www.gastrojournal. org), with lower doses (100 to 300 ␮g/kg) (Figure 3B), with administration as long as 48 hours before IR (Figure 3A), and with administration during reperfusion, that is, at 10 minutes or up to 1 hour after removal of the vascular clamp (Figure 3C). The livers of mice that received Diannexin and were then subjected to 90-minute ischemia and reperfusion for 24 hours were histologically normal or showed only minimal areas of necrosis and no or negligible numbers of inflammatory cells (Figure 3D and Table 2). In contrast, the annexin V monomer (“control” protein) did not confer hepatoprotection to the same degree as Diannexin (see Supplemental Figure 6B online at www.gastrojournal.org) at 24-hour reperfusion, as shown by serum ALT. This diminished effect of the monomer is likely due to its short half-life in the circulation (20 minutes).

Diannexin Protects Against Surface Injury to Hepatic SECs During Postischemic Reperfusion To challenge our hypothesis that surface damage to SECs plays a central role in the pathogenesis of IRI, we performed ultrastructural studies of the hepatic microvasculature in the same murine model, with or without Diannexin administration 5 minutes before applying the vascular clamp (that is, the identical procedure as in our whole animal studies). At 30 minutes of reperfusion, both SEM and TEM showed formation of gaps in SECs (Figure 4A and 4B). At 120-minute reperfusion, these changes were more pronounced. Administration of Diannexin (1 mg/kg IV) 5 minutes prior to prolonged ischemia near completely preserved the integrity of SECs and minimized gap formation (Figure 4C and 4D).

BASIC–LIVER, PANCREAS, AND BILIARY TRACT

August 2007

Figure 4. Diannexin preserves sinusoidal endothelial cell integrity in IRI. (A) Scanning electron micrograph (SEM) of sinusoid from a single mouse exposed to 90-minute ischemia and 2-hour reperfusion (original magnification, 17,500⫻). There are no normal sieve plates, and gaps in the plasma membrane are evident (arrows). (B) Transmission electron micrograph (TEM) of sinusoid from a single mouse exposed to 90-minute ischemia and 2-hour reperfusion (original magnification, 17,500⫻). Breaches (or gaps) in the integrity of the SEC plasma membrane are indicated by arrows. (C) SEM of sinusoid from a single mouse pretreated with Diannexin 5 minutes prior to 90-minute ischemia and 2-hour reperfusion (original magnification, 17,500⫻). Sieve plate pores (indicated by asterisks) are evident, and the surface of this SEC appears normal. (D) TEM of sinusoid from a single mouse pretreated with Diannexin 5 minutes prior to 90-minute ischemia and 2-hour reperfusion (original magnification, 17,500⫻). The normal lace-like appearance of the SEC plasma membrane, penetrated only by sieve plate pores (indicated by asterisks), is clearly evident. Electron micrographs are representative of n ⫽ 3 mice per experimental group. (E) Black and white photograph of hepatic sinusoid in naïve mouse subject to 90-minute ischemia and 2 hours of reperfusion. A swollen endothelial cell (EC) protrudes across more than 50% of the sinusoidal lumen (S). WBC, a leukocyte is adherent to the sinusoidal lining. The slowing of blood flow through the hepatic sinusoid at bottom left is evident by “stacked” red blood cells (RBC/arrow). (F) Black and white photograph of hepatic sinusoid (S) in Diannexin-treated mouse. Compared with naïve liver, there are no swollen hepatic ECs, no adherent leukocytes, and no stacking of red blood cells because of continuing rapid blood flow.

DIANNEXIN AND HEPATOPROTECTION

639

BASIC–LIVER, PANCREAS, AND BILIARY TRACT

August 2007

Figure 5. Diannexin prevents sinusoidal endothelial cell (EC) swelling, and microvascular recruitment of inflammatory cells and platelets. (A) Number of swollen SECs in hepatic sinusoids at 30 minutes or 120 minutes of postischemic reperfusion in periportal and centrilobular zones of the hepatic acinus. Experimental groups are vehicle-injected (Veh) or Diannexin-treated (D) (1 mg/kg 5 minutes prior to IR) sham-operated mice or animals subjected to 90-minute ischemia and 2-hour reperfusion. (B) Adherence of leukocytes in livers from the above experimental groups. #,ⴱP ⬍ .05 compared with vehicle controls. (C) Kupffer cell phagocytic activity (from ingestion of fluorescent latex beads) in livers from saline vehicle (Veh) controls and Diannexin-pretreated (D) mice subjected to 90-minute ischemia and 2-hour reperfusion. (D) Number of adherent platelets in hepatic sinusoids of controls (Veh) and Diannexin (1 mg/kg, 5 minutes prior to IR) (D) pretreated mice subjected to IR with reperfusion to 120 minutes, compared with sham-operated mice and animals subjected to 90-minute ischemia and 2-hour reperfusion. #,ⴱP ⬍ .05 compared with vehicle controls.

Diannexin Blocks Swelling of Hepatic SECs and Abolishes Inflammatory Cell Recruitment During Hepatic IRI Visualization of the hepatic microcirculation by in vivo microscopy allows direct observation of the dynamics of sinusoidal blood flow, impairment of which can be appreciated from the number of sinusoids in which flow is arrested.19 The sequence of events during hepatic IRI was gleaned by continuous observation of hepatic sinusoids by in vivo microscopy (see Supplemental Figure 7, video, online at www.gastrojournal.org). The dynamic

assessment indicated the following sequence of changes: SEC swelling (0 –30 minutes), KC activation, and leukocyte adherence to SECs (30 –120 minutes), followed by a gradual increase in platelet aggregation (90 minutes and beyond) as demonstrated by uptake of Rhodamine 6G30 (see Supplemental Figure 7, video, online at www. gastrojournal.org). Platelet aggregation was seen as circulating or adherent clumps of cells. As indicated, swollen SECs were conspicuous in both periportal and centrilobular regions within 30 minutes of postischemic reperfusion in the liver (Figures 4E and 5A).

640

TEOH ET AL

GASTROENTEROLOGY Vol. 133, No. 2

BASIC–LIVER, PANCREAS, AND BILIARY TRACT

Figure 5. (Cont’d.)

Administration of Diannexin (1 mg/kg IV) 5 minutes before clamping the hepatic blood vessels substantially reduced the number of swollen SECs in the periportal region at 30 minutes of reperfusion, and, by 120 minutes, the number of swollen cells was decreased by approximately 50% in both periportal and centrilobular zones (Figures 4F and 5A). Diannexin abolishes leukocyte activation and recruitment during hepatic IRI. Recruitment of circulating macrophages and other leukocytes contributes to inflammation within the microcirculation during IRI2,4,32,33; such cells are initially activated then adhere to the SECs that line the microcirculatory subunits. During the first 30 minutes of reperfusion, we observed very few or no leukocytes adherent to the SECs of the hepatic sinusoid (see Supplemental Figure 7 video, online at www.gastrojournal.org). However, during the next 90 minutes of reperfusion, leukocytes

attached to SECs with increasing regularity, as shown by the significantly increased number of adherent leukocytes at both 30 and 120 minutes of the reperfusion phase (Figure 5B). Diannexin appeared to reduce leukocyte adherence to the sinusoidal endothelium during the first 30 minutes (not significant) (Figures 4F and 5B) and, more impressively, prevented any further accumulation of adherent leukocytes up to 120 minutes (P ⬍ .05 or less) (Figure 5B). As shown in Figure 4F, this restored the hepatic microvasculature to apparently normal appearances. In the present work, we did not conduct detailed studies of KC activation, which has been shown by others to occur in early hepatic IRI.1,2,4 However, as determined from their physical appearance after ingestion of fluorescent-labelled latex particles, KC located in the periportal region (less so in centrilobular zones) were activated during IR, and Diannexin abolished such KC phagocytic activity (Figure 5C).

August 2007

DIANNEXIN AND HEPATOPROTECTION

641

Figure 6. Diannexin maintains sinusoidal blood flow during hepatic IRI. Number of perfused sinusoids in livers from saline vehicle controls (Veh) and Diannexin (1 mg/kg, 5 minutes prior to IR) pretreated mice (D), in sham-operated mice, and in animals subjected to 90-minute ischemia and 2-hour reperfusion. #,ⴱP ⬍ .05 compared with vehicle controls.

Diannexin appeared to substantially (60%–90%) prevent platelet aggregation (Figure 5D). At 120 minutes of reperfusion in Diannexin-treated mice, there was minimal evidence of platelet adherence either directly to the vascular endothelium or individually applied to attached leukocytes. In comparison, large clumps of adherent platelets were a prominent feature of microvascular injury in untreated mice at the corresponding period of IRI (see Supplemental Figure 5D and Figure 7 video, online at www.gastrojournal.org).

Diannexin Restores Sinusoidal Blood Flow During Hepatic IRI The effects of SEC injury and swelling, with resultant expression of proinflammatory mediators, leukocyte and platelet adherence, are to retard blood flow in the hepatic microcirculation. As shown during in vivo microscopy in naïve mice (see Supplemental Figure 7, video, online at www.gastrojournal.org), such changes in sinusoidal blood flow after IR were clearly evident by delayed passage of erythrocytes through narrowed sinusoids and intermittent blockage or reversal of sinusoidal flow, followed by its temporary restoration after clearance of adherent cells. In its more extreme manifestation, microvascular injury reduces the number of perfused sinusoids in the liver (Figure 6). In vehicle-treated control (naïve) mice subjected to hepatic IR, there were significant reductions (19%–28%) in the number of perfused periportal or centrilobular sinusoids at 30 and 120 minutes into the reperfusion phase. Diannexin substantially increased the number of perfused sinusoids in the centrilobular region after IRI and to a possibly lesser extent in the periportal region; these changes effectively restored hepatic microcirculatory blood flow to normal (see Supplemental Figure 7, video, online at www.gastrojournal.org).

Effects of Diannexin on Inflammatory Mediators During Hepatic IR ICAM-1 mediates the adhesion of leukocytes to activated endothelium via integrins, induces firm arrest of inflammatory cells at the vascular surface, and promotes leukocyte extravasation.34,35 VCAM-1 is transcriptionally induced on SECs but can also be expressed on macrophages and other cell types. Its interaction with integrins induces signals in endothelial cells that trigger changes in their morphology, thereby allowing leukocyte transmigration.36 Following 90minute ischemia and 2-hour reperfusion, there were 4-fold increases in hepatic messenger RNA (mRNA) levels of ICAM-1 (Figure 7A) and VCAM-1 (Figure 7B) compared with sham-operated mouse liver. Diannexin abrogated the changes in both ICAM-1 and VCAM-1 expression (Figure 7A and 7B). The real-time PCR findings were corroborated by Western immunoblotting of liver lysates, which revealed a striking increase in ICAM-1 protein expression in naïve liver subjected to IRI (Figure 7A). Likewise, immunohistochemistry for ICAM-1 in Diannexin pretreated liver showed a dramatic reduction in immunostaining compared with naïve liver injured by IRI (Figure 7C). MIP-2 is a 6-kilodalton heparin binding protein and C-X-C chemokine that exhibits potent chemotactic activity for inflammatory and immune effector cells of the monocyte/macrophage lineage.37 Diannexin significantly diminished expression of MIP-2 mRNA by 2-hour reperfusion compared with untreated liver (Figure 7D). Thromboxane A2 (TxA2) is another powerful stimulant of leukocyte recruitment and also exerts effects on platelet aggregation and vasoconstriction.38 Because it has a brief t1/2 of 37 seconds, production of TxA2 in vivo is determined by measuring its stable, nonenzymatic hydration product, TxB2. Using an ELISA to detect TxB2 in liver homogenates, we found that IRI triggered a marked

BASIC–LIVER, PANCREAS, AND BILIARY TRACT

Diannexin Inhibits Aggregation and Vascular Adherence of Platelets During Hepatic IRI

642

TEOH ET AL

increase in TxB2 production by 2-hour reperfusion, and levels of TxB2 were sustained at 24 hours (Figure 7E). Although Diannexin did not significantly affect early production of TxB2, by 24-hour reperfusion, the levels of this metabolite were substantially reduced (Figure 7E).

GASTROENTEROLOGY Vol. 133, No. 2

Although these findings suggest that Diannexin has an inhibitory effect on pathways involved with inflammatory and macrophage recruitment, it is also possible that the reduction in these inflammatory markers at 24-hour reperfusion may be the result of diminished injury in the

BASIC–LIVER, PANCREAS, AND BILIARY TRACT

Diannexin-treated animals, rather than inhibition of TxB2 formation per se.

Discussion Although ischemic preconditioning, conferred by one or more brief periods of clamping the hepatic blood flow, may protect against IRI caused by subsequent prolonged clamping,4,17 there are not yet any pharmacologic approaches that provide simpler, anticipatory, post hoc, or more effective protection against any form of IRI. Such an approach is needed because ischemic preconditioning (by vascular clamping) is clearly often not feasible to prevent reperfusion injury in nonsurgical contexts and is also ineffective after the onset of an ischemic event. Other strategies such as cytokine manipulation (eg, TNF-␣ blockade, interleukin-6 administration)1,2,18,39 and caspase inhibition40 have been shown to be hepatoprotective in animal models but are highly dose dependent, carry inherent possible adverse effects, and have no demonstrable efficacy when given after the onset of postischemic reperfusion. The present study in a murine model shows that Diannexin exerts potent and prolonged efficacy against hepatic IRI, a finding that is both highly novel41 and of potential clinical significance. Annexin V, a protein with anticoagulant activity, is known to exert antithrombotic effects by binding to PS. It has a short half-life by virtue of its small size (36 kilodaltons), which allows it to be rapidly cleared by the kidneys. Using recombinant DNA technology, we developed a homodimer of annexin V, called Diannexin, to exceed the renal filtration threshold by increasing its size (73 kilodaltons), thereby greatly prolonging its circulating and biologic half-life. The first studies with annexin V

DIANNEXIN AND HEPATOPROTECTION

643

effects and Diannexin were performed in human red blood cells; the results confirmed its higher affinity for externalized PS compared with the annexin V monomer, as well as its potent inhibitory activity on prothrombinase and phospholipase A2.28 Based on the high affinity of Diannexin for externalized PS residues28 and the facility of such residues to attract and activate leukocytes and platelets during the reperfusion phase, we anticipated that Diannexin would block inflammatory recruitment onto SECs, damage to which is known to be central to the pathogenesis of hepatic IRI,2,4 thereby compromising the hepatic microcirculation. Ultrastructural studies confirmed that Diannexin largely prevented SEC surface injury during the early stages of hepatic IR. Our limited study of KC phagocytosis is consistent with an additional or related effect of KC activation, although more detailed investigations are now required to establish the extent and mechanism of this effect. A role of KC activation is likely to be important in hepatic IR because the perisinusoidal location of this tissue macrophage allows it to release TNF-␣ and other proinflammatory molecules in response to microvascular injury,2,5 thereby augmenting leukocyte recruitment. Administration of Diannexin impressively reduced recruitment of activated leukocytes and platelets, which in the unprotected liver subjected to IRI can be observed to adhere to injured SECs and interrupt blood flow through hepatic sinusoids. The effects of Diannexin in reducing SEC swelling, inflammatory cell and platelet recruitment was associated with near complete preservation of sinusoidal structural integrity and significant improvement in sinusoidal blood flow. The kinetics, extent and duration of protection, and safety of Diannexin in these experiments all have practi-

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ Figure 7. Diannexin decreases vascular adherence and inflammatory mediators during hepatic IRI. (A) Hepatic ICAM-1 mRNA expression in mice subjected to IRI. 90/2R represents mice subjected to 90-minute ischemia, 2-hour reperfusion; D90/2R represents mice pretreated with Diannexin 1 mg/kg 5 minutes prior to 90-minute ischemia, 2-hour reperfusion; D10R denotes mice administered Diannexin 1 mg/kg at 10 minutes after onset of reperfusion; D60R denotes mice administered Diannexin 1 mg/kg at 60 minutes after onset of reperfusion. S, sham-operated mice. Results expressed as mean ⫾ SD for n ⫽ 3 mice per experimental group. *P ⬍ .05 compared with naïve (90/2R) mice. ICAM-1 immunoblots are from naïve and Diannexin-treated mice subjected to 90-minute ischemia, 24-hour reperfusion. Results are representative of n ⫽ 3 experiments performed in triplicate. Each lane contains 20 ␮g protein of liver homogenate from a single mouse at the indicated times of reperfusion. ␤-actin was used as the loading control. (B) Hepatic VCAM mRNA in mice subjected to IRI. Results are mean ⫾ SD for n ⫽ 3 mice per experimental group. *P ⬍ .001 compared with naïve (90/2R) mice. (C) Immunohistochemistry (IHC) for ICAM-1 was performed on livers from naïve and Diannexin-pretreated mice (original magnification, 200⫻). In naive mice subjected to hepatic IRI, perisinusoidal staining of ICAM-1 is evident at 2 hours, whereas minimal staining is seen in Diannexin-treated liver sections at the corresponding experimental time point. (D) MIP-2 mRNA expression in mice subjected to IRI. 90/2R represents mice subjected to 90-minute ischemia and 2-hour reperfusion; D90/2R represents mice pretreated with Diannexin (1 mg/kg) 5 minutes prior to 90-minute ischemia and 2-hour reperfusion; D10R denotes mice administered Diannexin (1 mg/kg) at 10 minutes after onset of reperfusion following 90-minute ischemia (analyzed at 24-hour reperfusion); D60R denotes mice administered Diannexin (1 mg/kg) at 60 minutes after onset of reperfusion following 90-minute ischemia (analyzed at 24 hours of reperfusion). (E) Hepatic thomboxane B2 levels determined by ELISA. 90/2R represents mice subjected to 90-minute ischemia, 2 hours of reperfusion; D90/2R represents mice pretreated with Diannexin (1 mg/kg) 5 minutes prior to 90-minute ischemia, 2-hour reperfusion; 90/24R represents mice subjected to 90-minute ischemia, 24 hours of reperfusion; D90/24R represents mice pretreated with Diannexin (1 mg/kg) 5 minutes prior to 90-minute ischemia, 24-hour reperfusion; 6D/24R represents mice given Diannexin (1 mg/kg) 6 hours prior to 90-minute ischemia, 24-hour reperfusion. S, sham-operated mice. Results expressed as mean ⫾ SD for n ⫽ 3 mice per experimental group.

BASIC–LIVER, PANCREAS, AND BILIARY TRACT

August 2007

644

TEOH ET AL

BASIC–LIVER, PANCREAS, AND BILIARY TRACT

cal implications for translation of these findings into clinical efficacy against hepatic IRI. Thus, whether administered 5 minutes up to 48 hours before or 1 hour after the onset of postischemic reperfusion, Diannexin provided extensive protection against hepatic IRI. Although not studied systematically in these experiments, Diannexin was remarkably free of postoperative complications, especially hemorrhage (data not shown, at least n ⫽ 60 animals). Furthermore, Diannexin provided extensive (⬎80%) protection against hepatocellular injury associated with hepatic IR. In the present work, protection against the early phase of hepatic IRI was shown by in vivo microscopy at 120 minutes of reperfusion, whereas protection against the later phase, when tissue inflammation is more conspicuous, was demonstrated at 24 hours of reperfusion by abrogation of ALT release and preservation of normal liver histology. Note that the murine model selected for use is not associated with mortality (which we find ethically unacceptable), and, therefore, survival benefit was not an aim demonstrated by these experiments. An important prediction from the present results is that Diannexin is likely to improve the preservation of organs for transplantation, thereby preventing the transplantation liver against early graft failure, a complication largely attributable to IRI.1 Although the mechanisms of warm and cold (preservation) injury differ in some details, injury to the hepatic SEC appears to play a central role in both types of injury.5,7,8 Based on the present results in hepatic IRI, Diannexin could also protect other organs, such as the intestine, heart, brain, and kidney, against the tissue damaging effects of reperfusion after prolonged ischemia, and this warrants further investigation. The critical role of circulating leukocytes and platelets in IRI indicated by the present results accords with earlier observations.1– 4,32,42 Thus, perfusion of livers preserved in vitro for 24 hours caused SEC apoptosis only when leukocytes or platelets were added, and synergistic effects were noted between these cell types. Roles for both polymorphonuclear leukocytes and monocytemacrophages have been demonstrated in hepatic IRI.32,33 Although it is possible that Diannexin exerts direct effects on leukocytes and platelets that explain its apparently diverse effects against hepatic microcirculatory damage in liver IRI, the present findings are equally consistent with the proposed central role of SEC injury. If the latter concept is correct, “capping” of everted PS residues by prolonged attachment of Diannexin would block PLA2 activation, which is a link between activation and attachment of leukocytes and platelets to the vascular endothelium that occurs some time after damage to the SEC. The time course of changes within the first 2 hours of IRI, as viewed by in vivo microscopy (see Supplemental Figure 7, video, online at www.gastrojournal.

GASTROENTEROLOGY Vol. 133, No. 2

org) is consistent with the latter observation. It is important to note that later inflammatory events within the liver subject to IRI are a response to earlier triggers that stimulate proinflammatory pathways such as adhesion molecule expression, chemokine and cytokine secretion, and PLA2 activation. The present data show that Diannexin causes near complete abrogation of the secondary phase of inflammatory injury. Thus, no or trivial numbers of leukocytes were evident in the liver at 24-hour post-IR after Diannexin administration, at a time when maximal inflammation was evident in unprotected livers.14,15,17,18 In summary, we tested whether a novel annexin V dimer, Diannexin, protects against hepatic IRI using a well-validated in vivo mouse model of moderately severe injury. As shown by ALT elevation, hepatic necrotic area, and liver inflammatory changes (24 hours), Diannexin provided ⬎85% protective efficacy. Importantly, this protective effect was conferred whether administered 5 minutes to 48 hours before IR, or up to 1 hour after reperfusion, indicating both prolonged and post hoc therapeutic efficacy. In conclusion, Diannexin is an apparently safe therapeutic protein that provides prolonged protection against hepatic IRI. The cellular mechanisms include apparent cytoprotection of SECs and interruption of secondary microcirculatory inflammation and coagulation.

Appendix Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.gastro. 2007.05.027. References 1. Banga NR, Homer-Vanniasinkam S, Graham A, Al-Mukhtar A, White SA, Prasad KR. Ischaemic preconditioning in transplantation and major resection of the liver. Br J Surg 2005;92: 528 –538. 2. Teoh N, Farrell GC. Hepatic ischemia reperfusion injury. Pathogenic mechanisms and basis for protection. J Gastroenterol Hepatol 2003;18:891–902. 3. Selzner N, Rudiger H, Graf R, Clavien PA. Protective strategies against ischemic injury of the liver. Gastroenterology 2003;125: 917–936. 4. Jaeschke H. Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning. Am J Physiol Gastrointest Liver Physiol 2003;284:G15–G26. 5. Samarasinghe DA, Tapner M, Farrell GC. Role of oxidative stress in hypoxia-reoxygenation injury in cultured rat hepatic sinusoidal endothelial cells. Hepatology 2000;31:160 –165. 6. Sindram D, Porte RJ, Hoffman MR, Bentley RC, Clavien PA. Synergism between platelets and leukocytes in inducing endothelial cell apoptosis in the cold ischemic rat liver: a Kupffer cell mediated injury. FASEB J 2001;15:1230 –1232. 7. Clavien PA. Sinusoidal cell injury during hepatic preservation and reperfusion. Falk Symp 2001;117:279 –287.

8. Natori S, Selzner M, Valentino KL, Fritz LC, Srinivasan A, Clavien PA, Gores GJ. Apoptosis of sinusoidal endothelial cells occurs during liver preservation injury by a caspase-dependent mechanism. Transplantation 1999;68:89 –96. 9. Devaux PF, Zachowsky A. Maintenance and consequences of membrane phospholipid asymmetry. Chem Phys Lipids 1994;73: 107–120. 10. Vajdova K, Graf R, Clavien PA. ATP supplies in the cold-preserved liver: a long neglected factor of organ viability. Hepatology 2002; 36:1543–1552. 11. Hammill AK, Uhr JW, Scheuermann RH. Annexin V staining due to loss of membrane asymmetry can be reversible and precede commitment to apoptotic cell death. Exp Cell Res 1999;251: 16 –21. 12. Ran S, Downes A, Thorpe PE. Increased exposure of anionic phospholipids on the surface of tumor blood vessels. Cancer Res 2002;62:6132– 6140. 13. Colletti LM, Remick DG, Burtch GD, Kunkel SL, Strieter RM, Campbell DA Jr. Role of tumor necrosis factor-␣ in the pathophysiologic alterations after hepatic ischemia/reperfusion injury in the rat. J Clin Invest 1990;85:1936 –1943. 14. Rudiger HA, Clavien P. Tumor necrosis factor ␣, but not Fas, mediates hepatocellular apoptosis in the murine ischemic liver. Gastroenterology 2002;122:202–210. 15. Teoh N, Field J, Sutton J Farrell G. Dual role of tumor necrosis factor-␣ in hepatic ischemia reperfusion injury: studies in TNF-␣ knockout mice. Hepatology 2004;39:412– 421. 16. Husted TL, Lentsch AB. The role of cytokines in pharmacological modulation of hepatic ischemia/reperfusion injury. Curr Pharm Des 2006;12:2867–2873. 17. Teoh N, Dela Pena A, Farrell G. Hepatic ischemic preconditioning in mice is associated with activation of NF-␬B, p38 kinase, and cell cycle entry. Hepatology 2002;36:94 –102. 18. Teoh N, Leclercq I, dela Pena A, Farrell G. Low-dose TNF-␣ is protective against hepatic ischemia-reperfusion injury in mice: mechanistic implications for preconditioning. Hepatology 2003; 37:118 –128. 19. McCuskey RS. Microscopic methods for studying the microvasculature of internal organs. In: Baker CH, Nastuk WT, eds. Physical techniques in biology and medicine microvascular technology. New York: Academic Press, 1986:247–264. 20. Dela Pena A, Leclercq I, Field J, Jones B, Farrell, G. NF-␬B activation, rather than TNF, mediates hepatic inflammation in a murine dietary model of steatohepatitis. Gastroenterology 2005; 129:1663–1674. 21. Majumder R, Wang J, Lentz BR. Effects of water soluble phosphotidylserine on bovine factor Xa: functional and structural changes plus dimerization. Biophys J 2003;84:1238 –1251. 22. Reutelingsperger CPM, Hornstra G, Hemker HC. Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord. Eur J Biochem 1985;151:625– 629. 23. Harris EN, Pierangeli SS. Functional effects of anticardiolipin antibodies. Lupus 1996;5:372–377. 24. Romisch J, Seiffge D, Reiner G, Paques EP, Heimburger N. In vivo antithrombotic potency of placenta protein 4 (annexin V). Thromb Res 1991;61:93–104. 25. Van Ryn-McKenna J, Merk H, Muller TH, Buchanan MR, Eisert WG. The effects of heparin and annexin V on fibrin accretion after injury in the jugular vein in rabbits. Thromb Haemost 1993;69: 227–230. 26. Thiagarajan P, Benedict CR. Inhibition of arterial thrombosis by recombinant annexin V in a rabbit carotid artery injury model. Circulation 1997;96:2339 –2347. 27. Freyssinet JM, Toti-Orfanoudakis F, Ravanat C, Grunebaum L, Gauchy J, Cazenave JP, Wiesel ML. The catalytic role of anionic phospholipids in the activation of protein C by factor Xa and

DIANNEXIN AND HEPATOPROTECTION

28.

29.

30.

31.

32.

33.

34. 35.

36.

37.

38.

39.

40.

41.

42.

645

expression of its anticoagulant function in human plasma. Blood Coagul Fibrinolysis 1991;2:691– 698. Kuypers FA, Larkin LK, Emerson RK, Allison AC. Di-Annexin, a novel tool to identify and protect phosphatidylserine exposing cells. Blood 2004;104:441a. Billy D, Speijer H, Zwaal RF, Hack EC, Hermens WT. Anticoagulant and membrane-degrading effects of secretory (non-pancreatic) phospholipase A2 are inhibited in plasma. Thromb Haemost 2002;87:978 –984. Fourcade O, Simon MF, Viode C, Rugani N, Leballe F, Ragab A, Fournie B, Sarda L, Chap H. Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells. Cell 1995;80: 919 –927. Okusa MD, Ye H, Huang L, Sigismund L, Macdonald T, Lynch KR. Selective blockade of lysophosphatidic LPA3 receptors reduces murine renal ischemia-reperfusion injury. Am J Physiol Renal Physiol 2003;285:F565–F574. Jaeschke H, Farhood A, Smith CW. Neutrophils contribute to ischemia-reperfusion injury in rat liver in vivo. FASEB J 1990;4: 3355–3359. Jaeschke H, Farhood A, Bautista AP. Complement activates Kupffer cells and neutrophils during reperfusion after hepatic ischemia. Am J Physiol Gastrointest Liver Physiol 1993;264: G801–G809. Libby P, Rodker PM, Maseri A. Inflammation and atherosclerosis. Circulation 2002;105:1135–1143. Leeuwenberg JF, Smeets EF, Neefjes JJ, Shaffer MA, Cinek T, Jeunhomme TM, Ahern TJ, Buurman WA. E-selectin and intercellular adhesion molecule-1 are released by activated human endothelial cells in vitro. Immunology 1991;77:543–549. Matheny HE, Deem TL, Cook-Mills JM. Lymphocyte migration through monolayers of endothelial cell lines involves VCAM-1 signaling via endothelial cell NADPH oxidase. J Immunol 2000; 164:6550 – 6559. Driscoll KE, Hassenbein DG, Howard BW, Isfort RJ, Cody D, Tindal MH, Suchanek M, Carter JM. Cloning, expression and functional characterization of rat macrophage inflammatory protein 2: a neutrophil chemoattractant and epithelial cell mitogen. J Leuk Biol 1995;58:359 –364. Allman-Farinelli MA, Dawson B. Diet and aging: bearing on thrombosis and hemostasis. Semin Thromb Hemost 2005;31:111– 117. Teoh N, Field J, Farrell G. Interleukin-6 is a key mediator of the hepatoprotective and pro-proliferative effects of ischemic preconditioning in mice. J Hepatol 2006;45:20 –27. Hoglen NC, Anselmo DM, Katori M, Kaldas M, Shen X-D, Valentino KL, Lassman C, Busuttil RW, Kupiec-Weglinski JW, Farmer DG. A caspase inhibitor, IDN-6556, ameliorates early hepatic injury in an ex vivo rat model of warm and cold ischemia. Liver Transpl 2007;13:361–366. Teoh N, Ito Y, Field J, McCuskey M, McCuskey R, Farrell GC, Allison AC. Diannexin, a novel Annexin V homodimer, provides prolonged microvascular protection against warm hepatic ischemia-reperfusion injury in mice. Hepatology 2005;42:314A. Hamamoto I, Maeba T, Tanaka S. Role of leukocytes during the early phase of reperfusion injury after cold preservation of rat liver. Ann N Y Acad Sci 1999;723:476 – 479.

Received January 4, 2007. Accepted May 3, 2007. Address requests for reprints to: Geoffrey C. Farrell, MD, FRACP, Professor of Hepatic Medicine and Director of Gastroenterology, Gastroenterology and Hepatology Unit, Australian National University Medical School at The Canberra Hospital, Level 2, Building 1, Yamba Drive, Garran, ACT 2605 Australia. e-mail: [email protected]; fax: (61) 2 62815179.

BASIC–LIVER, PANCREAS, AND BILIARY TRACT

August 2007

646

TEOH ET AL

Supported by project grant 211137 and program grant 358398 of the Australian National Health and Medical Research Council (NHMRC) as well as the Robert W. Storr Bequest of the University of Sydney Medical Foundation for research in the Storr Liver Unit and supported in part by the National Institutes of Health NIH/NIAAA grant RO1 AA12436. N.T. was an Australian CJ Martin NHMRC and American Liver Foundation Postdoctoral Research Fellow. The authors thank the colleagues who contributed to this research program in different ways: G. Ringold for support throughout the

GASTROENTEROLOGY Vol. 133, No. 2

Diannexin program. P. Thiagarajan for helpful input. H. Schulman for contribution to design of Diannexin, which was produced in Escherichia coli by L. Kakach and J. Malinski. F. Kuypers, S. Larkin, and J. Emeis and their colleagues for demonstrating the antithrombotic activity of Diannexin and its effect on PLA2. Financial disclosure: A. Allison is an employee and shareholder of Alavita. None of the other authors have competing financial interests, with the exception of partial reimbursement of research expenses.

BASIC–LIVER, PANCREAS, AND BILIARY TRACT