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Keywords:

  • Adhesion molecules;
  • ischemia/reper-fusion injury;
  • liver transplantation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Ischemia/reperfusion injury (IRI) remains an important problem in clinical transplantation. Following ischemia, phosphatidylserine (PS) translocates to surfaces of endothelial cells (ECs) and promotes the early attachment of leukocytes/platelets, impairing microvascular blood flow. Diannexin, a 73 KD homodimer of human annexin V, binds to PS, prevents attachment of leukocytes/platelets to EC, and maintains sinusoidal blood flow. This study analyzes whether Diannexin treatment can prevent cold IRI in liver transplantation. Rat livers were stored at 4°C in UW solution for 24 h, and then transplanted orthotopically (OLT) into syngeneic recipients. Diannexin (200 μg/kg) was infused into: (i) donor livers after recovering and before reperfusion, (ii) OLT recipients at reperfusion and day +2. Controls consisted of untreated OLTs. Both Diannexin regimens increased OLT survival from 40% to 100%, depressed sALT levels, and decreased hepatic histological injury. Diannexin treatment decreased TNF-α, IL-1β, IP-10 expression, diminished expression of P-selectin, endothelial ICAM-1, and attenuated OLT infiltration by macrophages, CD4 cells and PMNs. Diannexin increased expression of HO-1/Bcl-2/Bcl-xl, and reduced Caspase-3/TUNEL+ apoptotic cells. Thus, by modulating leukocyte/platelet trafficking and EC activation in OLTs, Diannexin suppressed vascular inflammatory responses and decreased apoptosis. Diannexin deserves further exploration as a novel agent to attenuate IRI, and thereby improve OLT function/increase organ donor pool.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Despite improvements in organ preservation, surgical techniques and immunosuppressive regimes, post-ischemic reperfusion injury (IRI), remains an important problem in clinical organ transplantation. IRI contributes to both early and late dysfunction of liver grafts (1–3). Moreover, IRI exacerbates the shortage of donor livers because of the higher susceptibility of marginal organs to the ischemic insult (4). Minimizing the adverse effects of IRI could significantly increase the number of donor organs, allowing more patients to be successfully transplanted.

The development of a therapeutic agent to attenuate IRI requires an understanding of its pathogenesis, which involves a complex interaction of several factors. These are largely Ag-independent, although IRI can augment immune responses to allo-Ag in organ grafts (5). Studies carried out during the past decade have provided clues to the pathogenesis of IRI in the liver, including activation of Kupffer cells, the release of pro-inflammatory cytokines, increased expression of adhesion molecules, leukocyte infiltration and microthrombus formation (3,6–8). Most relevant for the development of an efficacious therapy are early events in IRI; by inhibiting them it may be possible to decrease the severity of downstream consequences that result in vascular and end-organ damage.

Early stages of warm IRI in the mouse liver have been revealed by the intravital microscopic studies of Teoh et al. (9). In the sinusoids of the normal liver endothelial cells (ECs) are flat, and blood flow is rapid. In contrast, during the period 1–3 h of postischemic reperfusion EC become rounded and partially occlude the lumina of sinusoids. During the same critical period leukocytes and platelet aggregates adhere to EC and further impede sinusoidal blood flow, which is almost completely stopped. These early events trigger later stages of IRI, which result in ultimate hepatocyte damage.

A major early event in the pathogenesis of warm murine liver IRI is externalization of phosphatidylserine (PS) in sinusoidal ECs as a result of anoxia (10) and reoxygenation (11). A recombinant homodimer of human annexin V, termed Diannexin (12), was used to bind PS on cell surfaces, thereby suppressing the attachment of leukocytes and platelet aggregates to ECs. Diannexin was found to have these predicted effects and to preserve sinusoidal blood flow in the mouse liver during postischemic reperfusion (9). Consequently, hepatocyte injury 24 h later was markedly decreased, as shown by histology and serum levels of alanine aminotransferase. These findings suggested that Diannexin may also prevent cold IRI associated with liver transplantation. Here, we present our findings in a well-characterized model in which rat livers are stored in the cold before orthotopic transplantation into syngeneic recipients (OLT). Diannexin was found to prevent IRI, suggesting that it may be useful in human organ transplantation. Mechanisms by which Diannexin exerts these potent desirable effects were analyzed, and the findings are reviewed in the discussion.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Animals

Male Sprague–Dawley (SD) rats weighing 230–280 g (Harlan Sprague–Dawley, Inc., San Diego, CA) were maintained under conditions approved by the UCLA Chancellor's Animal Research Committee. All animals were housed in micro-isolator cages in virus-free facilities and fed laboratory chow ad libitum.

Liver IRI model and diannexin treatment

The SD livers were stored at 4°C in UW solution for 24 h prior to being transplanted into syngeneic SD recipients with revascularization but without hepatic artery reconstruction, as described (13). There were four experimental groups: Gr. I: donor livers were perfused ex vivo with Diannexin (Alavita Inc., Mountain View, CA; 200 μg/kg b.w. via portal vein) after recovering and immediately before reperfusion. Gr. II: OLT recipients were infused with Diannexin (200 μg/kg b.w. i.v.) immediately after liver reperfusion and 2 days after transplantation. Gr. III: untreated OLT controls, and Gr. IV: sham controls.

Hepatocellular function assay

Serum alanine aminotransferase (sALT) levels, indicator of hepatocellular injury, were measured using an auto analyzer (ANTECH Diagnostics, Los Angeles, CA).

Myeloperoxidase assay

The presence of myeloperoxidase (MPO), an enzyme specific for neutrophils, was used as an index of PMN accumulation in the liver (14). Briefly, the frozen tissue was thawed and placed in 4 mL iced 0.5% hexadecyltrimethylammonium bromide and 50 mmol potassium phosphate buffer solution with the pH adjusted to 5. Each sample was homogenized for 30 s and centrifuged at 15 000 rpm for 15 min at 4°C. Supernatants were then mixed with hydrogen peroxide-sodium acetate and tetramethylbenzidine solutions. The change in absorbance was measured spectrophotometrically at 655 nm. One unit of MPO activity was defined as the quantity of enzyme degrading 1 μmol peroxide/min at 25°C/g of tissue.

Histology and immunohistochemistry

Liver specimens were fixed in 10% buffered formalin and embedded in paraffin. Liver sections (4 μm) were stained with hematoxylin/eosin, and then analyzed blindly. The histological severity of IRI was graded using Suzuki's criteria (7), with modifications. In this classification, sinusoidal congestion, hepatocyte necrosis and ballooning degeneration are graded from 0 to 4. No necrosis, congestion/centrilobular ballooning is given a score of 0, whereas severe congestion/ballooning degeneration, as well as >60% lobular necrosis is given a value of 4.

OLT samples were examined by immunohistochemistry for endothelial ICAM-1 expression as well as monocyte/macrophage and CD4+ T-cell infiltration, as described (15,16,17). Briefly, liver tissue was embedded in Tissue Tec OCT compound (Miles, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at −70°C. Cryostat sections (5 μm) were fixed in acetone and then endogenous peroxidase activity was inhibited with 0.3% H2O2 in PBS. Primary mouse antibodies against rat ICAM-1 (1A29), RecA-1, a pan-endothelial cell marker (HIS52), monocytes/macrophages (ED1) or CD4+ (OX-35) were added at optimal dilutions (BD Biosciences Pharmingen, San Jose, CA). Bound primary antibody was detected using biotinylated anti-mouse IgG and streptavidin peroxidase-conjugated complexes (DAKO, Carpinteria, CA). Negative controls included sections in which the primary antibody was replaced with either dilution buffer or normal mouse serum. The sections were evaluated blindly by counting the labeled cells in triplicates or assessing the intensity of staining (for ICAM) within 10 high-power fields per section.

RNA extraction and competitive template RT-PCR

To study cytokine gene expression patterns, we used competitive template reverse transcription-polymerase chain reaction (RT-PCR), as described (18). Briefly, total RNA was extracted from frozen liver tissue samples, using an RNase minikit (Qiagen, Chatsworth, CA), and RNA concentration was determined with a spectrophotometer. Five micrograms of RNA was reverse transcribed, using oligo(dT) primers and SuperScript reverse transcriptase (GIBCO, Carlsbad, CA). According to the varying contents of specific cDNA and amplification efficiencies, PCR was performed with different cycle numbers at the annealing temperature that was optimized empirically for each primer pair: 35 cycles at 60°C (TNF-α), 35 cycles at 60°C (IL-1β), 35 cycles at 68°C (IL-6), 35 cycles at 53°C (IP-10), 35 cycles at 58°C (ICAM-1), 35 cycles at 58°C (P-selectin), and 35 cycles at 63°C (β-actin), respectively. PCR products were analyzed in ethidium bromide-stained 2% agarose gel, and the density was scanned with Kodak Digital Science 1D image analysis software (version 2.0; Eastman Kodak Scientific Imaging Systems, New Haven, CT). To compare relative levels of each cytokine in different samples, all samples were normalized against the respective β-actin template cDNA ratio.

Detection of apoptosis

A commercial histochemical assay (Klenow-FragEL, Oncogene Research Products, Cambridge, MA) was used to detect DNA fragmentation in cryostat OLT samples (17). Biotinylated nucleotides were detected using streptavidin-horse radish peroxidase (HRP) conjugate. Counter-staining with methyl green aids in the morphological evaluation and characterization of normal versus apoptotic cells. Results were scored semi-quantitatively by averaging the number of apoptotic cells/microscopic field at 200× magnification. Ten fields were evaluated/tissue sample.

Western blot analysis

Protein was extracted from tissue samples with protein lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate and 1% Triton X-100, pH 7.2). Proteins (30 μg/sample) in SDS-loading buffer (50 mM Tris, pH 7.6, 10% glycerol, 1% SDS) were subjected to 12% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA). The gel was then stained with Coomassie blue for protein loading. The membrane was blocked with 3% dry milk and 0.1% Tween 20 (USB, Cleveland, OH) in PBS. Polyclonal rabbit anti-rat HO-1 Ab (Stressgen, Victoria, BC, Canada), rabbit anti-rat Bcl-2/Bcl-xl and anti-rat Caspase-3 Abs (Santa Cruz Biotechnology, Santa Cruz, CA) were used. The membranes were incubated with Abs and developed according to the Amersham Enhanced Chemiluminescence protocol. Relative quantities of HO-1, Bcl-2/Bcl-xl, and Caspase-3 proteins were determined by densitometer (Kodak Digital Science 1D Analysis Software).

Statistical analysis

All values are expressed as mean ± SD. Animal survival was evaluated by Kaplan–Meier method and the log-rank test. Data were analyzed with an unpaired 2-tailed Student t test. The p-value of <0.05 was considered to be statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Diannexin treatment protects against cold IRI in the rat OLT model

To test the efficacy of Diannexin treatment against hepatic IRI, we used a well-established rat liver model of 24 h cold ischemia, followed by OLT. As shown in Figure 1A only 40% of untreated recipients (4/10) remained alive at day +14, with the majority of death occurring within the first 2 posttransplant days. In marked contrast, 100% of OLT recipients (10/10; p < 0.05) were alive at day +14 after ex vivo perfusion of donor livers with Diannexin or following treatment of OLT recipients with Diannexin.

imageimageimage

Figure 1. (A) OLT survival. SD rat livers were stored at 4° C in UW solution for 24 h prior to transplantation to syngeneic SD recipients. (♦), donor livers were perfused with Diannexin (200 μg/kg b.w. via portal vein) after recovering and immediately before liver reperfusion; (▪), OLT recipients were given Diannexin (200 μg/kg b.w. i.v.) immediately after liver reperfusion and at day +2 posttransplant; (▴), untreated controls. Untreated OLT survival at 14 days was 40% vs. 100% in Diannexin-treated groups (n = 10 rats/groups; p < 0.05). (B) sALT levels in OLT recipients. SD livers were recovered and stored at 40 C in UW solution for 24 h, and then transplanted to syngeneic rats. Serum samples were taken at 6 h, 1 day and 3 days after reperfusion. sALT levels were markedly lower in Diannexin-treated donor (hatched bars) and recipient (white bars) groups at 6 h, 3 days (*p < 0.05) and 1 day (***p < 0.001) after reperfusion, as compared with controls. N = 4–6 experiments/group. Mean and SD are shown. (C) Liver histology at 6 h and 1 day posttransplant. A & C, untreated OLTs with prominent sinusoidal/vascular congestion and lobular ballooning with 15–30% of hepatocyte necrosis (Suzuki score: 3.0 ± 0.7 and 3.2 ± 1.1, respectively); B & D, Diannexin-treated OLT recipients with minimal necrosis/sinusoidal congestion and good preservation of lobular architecture (Suzuki score 1.0 ± 0.41 and 1.1 ± 0.6, respectively, p < 0.01) (magnification × 100, H&E stain; representative of n = 4/group).

Prolonged OLT survival after Diannexin treatment correlated with markedly improved hepatocellular function (Figure 1B). After 24 h cold preservation followed by OLT, sALT levels (IU/L) in Diannexin-treated donor or recipient group were significantly decreased as compared with untreated controls (6 h: 600 ± 35, 267 ± 110 vs. 1345 ± 530, p < 0.05; 1 day: 481 ± 238, 460 ± 194 vs. 4031 ± 383, p < 0.001; 3 days: 89 ± 48.9, 65 ± 8 vs. 169 ± 49, p < 0.05).

These functional data correlated with histological Suzuki's grading of hepatic injury. As shown in Figure 1C, untreated group revealed prominent sinusoidal and vascular congestion as well as lobular ballooning with 15–30% of hepatocyte necrosis at 6 h and day 1 after reperfusion. (Figure 1A and C; Suzuki score, 3.0 ± 0.7 and 3.2 ± 1.1, respectively). In contrast, OLTs from Diannexin-treated recipients showed minimal necrosis/sinusoidal congestion, and good preservation of lobular architecture (Figure 1B and D; Suzuki score 1.0 ± 0.41 and 1.1 ± 0.6, respectively; p < 0.01).

Diannexin depresses chemokine/adhesion molecule expression and endothelial activation in OLTs

We assessed mRNA levels coding for IP-10, P-selectin and ICAM-1 that mediate early adhesive interactions between leukocytes and endothelium at the inflammatory sites. As shown in Figure 2A, Diannexin-treatment markedly attenuated OLT expression of IP-10 (6 h: 0.79 ± 0.26 vs. 1.85 ± 0.15; 1 day: 1.21 ± 0.22 vs. 2.23 ± 0.18; 3 days: 0.65 ± 0.12 vs. 1.66 ± 0.13; p < 0.05), P-selectin (6 h: 0.55 ± 0.16 vs. 1.34 ± 0.14; 1 day: 1.35 ± 0.15 vs. 2.11 ± 0.18; 3 days: 0.41 ± 0.14 vs. 1.15 ± 0.16; p < 0.05), and ICAM-1 (6 h: 0.67 ± 0.16 vs. 1.42 ± 0.11; 1 day: 1.1 ± 0.19 vs. 1.95 ± 0.15; 3 days: 0.52 ± 0.16 vs. 1.15 ± 0.09; p < 0.05) after reperfusion, as compared with untreated controls.

imageimage

Figure 2. (A) Competitive template RT–PCR-assisted OLT detection of chemokines and adhesive molecules. Treatment of OLT recipients with Diannexin (white bars) diminished the expression of IP-10, P-selectin and ICAM-1 at 6 h, day 1 and day 3 after posttransplant (*p < 0.05), as compared with untreated control (black bars). Each column represents the mean ± SD of 4–5 independent experiments. (B) Immunopathology of rat sinusoidal endothelium in OLTs exposed to cold ischemia (24 h) followed by reperfusion. Immunostaining for ICAM-1 (A, B, E and F) and RecA-1, a pan-endothelial marker (C, D, G and H) in livers recovered at 6 h (A, E, C, G) and 1 day (B, F, D and H) after transplantation from Diannexin-treated (A–D) and untreated (E–H) controls. Data are representative of three recipients/group (immunoperoxidase labeling of cryostat sections, magnification ×200).

To directly test the effects of Diannexin treatment upon the activation of sinusoidal endothelium in OLTs, we then analyzed ICAM-1 expression by immunohistology. Figure 2B shows immunostaining for ICAM-1 (A, B, E and F), and RecA-1 (C, D, G, and H) in livers recovered at 6 h (A, E, C, G) and 1 day (B, F, D and H) after OLT from Diannexin-treated rats (A–D) and respective controls (E–H). Indeed, ICAM-1 expression was reduced in Diannexin-treated livers as compared to respective control OLTs (+ vs. ++), providing an indication that Diannexin depressed endothelial cell activation. In contrast, the expression of RecA-1, a pan-endothelial cell marker, was strongly detected (+++) in all studied specimens.

Diannexin decreases leukocyte traffic in OLTs

To determine whether Diannexin affected OLT leukocyte infiltration, we analyzed OLTs for PMN infiltration by MPO assay and for macrophage (ED-1)/CD4 T-cell (0X-35) infiltration by immunohistochemical staining. Indeed, as shown in Figure 3A, MPO activity in Diannexin-treated OLT group was significantly decreased at 6 h (1.73 ± 0.22 vs. 3.81 ± 0.23, p < 0.05), at 24 h (2.14 ± 0.25 vs. 4.7 ± 0.32, p < 0.05) and at 3 days (0.75 ± 0.14 vs. 1.56 ± 0.14, p < 0.05) after transplantation, as compared with untreated control group. Moreover, Diannexin therapy dramatically diminished OLT infiltration by macrophages (Figure 3B; 6 h: 6.1 ± 0.57 vs. 15.5 ± 1.9/HPF; p < 0.01; 1 day: 7.75 ± 0.64 vs. 14.13 ± 0.97/HPF; p < 0.01) and CD4+ T cells (Figure 3C; 6 h: 6.3 ± 2.3 vs. 21.2 ± 6.2; p < 0.05; 1 day: 6.2 ± 0.7 vs. 18.7 ± 2.1, p < 0.05) following reperfusion, compared with untreated control OLTs.

imageimageimage

Figure 3. (A) MPO-assisted detection of neutrophil enzymatic activity (U/gm) in liver tissue. Treatment of OLT recipients with Diannexin (white bars) decreased MPO activity at 6 h, 1 day and 3 days after reperfusion, compared with untreated control (black bars; *p < 0.05). These data represent 4–5 independent experiments. Means and SD are shown. Immunohistochemical staining of OLTs at 6 h and 1 day posttransplant. (B) ED-1+ macrophages; (C) OX-35+ CD4 T cells. Diannexin recipient treatment (white bars) markedly reduced OLT cell infiltration, compared with untreated control (black bars; macrophages: **p < 0.01; CD4 T cells: *p < 0.05). Means and SD are shown (n = 3–5 rats/group). Representative staining of 3 OLTs/group are shown. (Original magnification × 400.)

Diannexin reduces OLT pro-inflammatory cytokine expression

We used competitive template RT-PCR to analyze cytokine gene expression in OLTs. As shown in Figure 4, Diannexin treatment markedly decreased intragraft expression of mRNA coding for TNF-α, IL-1β and IL-6 throughout the observation period (6 h, 1 day and 3 days after reperfusion), as compared with control OLTs (p < 0.05).

image

Figure 4. Competitive template RT-PCR-assisted expression of pro-inflammatory cytokines at 6 h, day 1 and day 3 after OLT. Recipient treatment with Diannexin (white bars) significantly reduced the expression of TNF-α, IL-1β and IL-6 (*p < 0.05), compared with untreated controls (black bars). Each column represents the mean ± SD of four to five independent experiments.

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Diannexin depresses apoptosis and up-regulates cytoprotective molecules in OLTs

We used TUNEL assay to examine apoptosis events in OLTs. As shown in Figure 5A, Diannexin treatment significantly reduced the frequency of TUNEL+ cells after reperfusion, as compared with untreated group (6 h: 3.07 ± 0.87 vs. 13.03 ± 1.93; 1 day: 3.6 ± 1.15 vs. 22.33 ± 3.72, p < 0.01).

imageimage

Figure 5. (A) TUNEL-assisted detection of apoptosis in OLTs at 6 h and 1 day. Upper panel: Apoptotic cells in Diannexin-treated recipient group (white bars) were diminished after reperfusion, as compared with untreated group (black bars; **, p < 0.01). Mean and SD are shown (n = 3–5 rats/group). Lower panels: Representative staining of apoptotic positive cells in ischemic livers at I day after reperfusion (Original magnification × 400). (B) Western blot analysis of OLT expression of antioxidant (HO-1), antiapoptotic (Bcl-2/Bcl-xl) and pro-apopototic (Caspase-3) proteins. The expression of HO-1, Bcl-2, Bcl-xl and Caspase-3 was probed by employing Abs against rat (A) HO-1, (B) Bcl-2, (C) Bcl-xl, (D) Caspase-3. Lane1-sham group; Lane 2-, 3- and 4-control group (6 h, 1 day and 3 days, respectively); Lane 5-, 6- and 7- Diannexin-treated recipient group (6 h, 1 day and 3 days, respectively). Diannexin treatment significantly increased expression of HO-1, Bcl-2/Bcl-xl, compared with untreated group. However, the expression of Caspase-3 in the treatment group was inhibited, compared with control. The data are representative of four separate experiments.

Then, we used Western blots to evaluate the expression of anti-oxidant (HO-1), anti-apoptotic (Bcl-2/Bcl-xl), and pro-apoptotic (Caspase-3) gene products. The relative intragraft expression levels in absorbance units (AU) were analyzed by densitometry. As shown in Figure 5B, Diannexin treatment strongly up-regulated the expression of HO-1 (lane 5–7 A, respectively: 1.8–2.1 AU) and Bcl-2/Bcl-xl (lane 5–7 B, respectively; 1.6–1.9 AU and C, respectively; 1.5–1.8 AU) but inhibited the levels of Caspase-3 (lane 5–7 D, respectively; 0.7–0.9 AU) at 6 h, 1 day and 3 days after OLTs. In contrast, the corresponding OLT samples in untreated group revealed much enhanced Caspase-3 levels (lane 2–4 D, respectively; 1.8–2.0 AU) and markedly decreased expression of HO-1 (lane 2–4 A, respectively; 0.3–0.4 AU) and Bcl-2/Bcl-xl (lane 2–4 B, respectively; 0.1–0.3 AU and C, respectively; 0.2–0.4 AU).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

We report here the results of our studies on the effects of Diannexin, a recombinant human annexin V homodimer, in a rat liver model of 24 h cold ischemia followed by transplantation. Two Diannexin schedules of ex vivo liver perfusion or treatment of OLT recipients: (1) improved liver function/hepatocyte integrity, with resultant prolongation of graft survival; (2) decreased endothelial cell activation and the expression of adhesion molecules required for leukocyte recruitment; (3) prevented macrophage/T cell/neutrophil sequestration; (4) inhibited the expression of pro-inflammatory cytokines; (5) augmented the expression of anti-apoptotic/anti-oxidant genes and (6) reduced hepatic apoptosis. Although Diannexin exerts anti-thrombotic activity we did not observe increased hemorrhage or additional bleeding. This is consistent with the minimal impact of annexin V or Diannexin on systemic hemostasis (12,19–20). Thus, Diannexin represents a new generation of therapeutic agents in organ transplantation. By inhibiting early IRI events, Diannexin blocks a cascade of subsequent events that promote recruitment of leukocytes, impair microvascular blood flow and produce end-organ damage. This therapeutic intervention may prove to be more effective than attempts to inhibit individual later events, such as leukocyte recruitment, the formation of inflammatory mediators or the action of caspases in apoptosis.

Diannexin was developed to meet the need for an agent that binds PS on cell surfaces with high affinity and survives long enough in the circulation to exert useful therapeutic activity. The human protein annexin V has four sites binding PS in a Ca2+-dependent manner (21) and is routinely used as an agent to detect PS externalization in the course of apoptosis (22). However, the 36 KD annexin V monomer passes rapidly from the circulation into the urine; the t 1/2 in the circulation is 20 min or less (19). In contrast, the recombinant homodimer of human annexin V, Diannexin (73KD), exceeds the renal filtration threshold and is retained in the circulation long enough for desired therapeutic activities (t 1/2 > 2 h); additionally, Diannexin has a higher affinity for PS on cell surfaces than the monomer does (12).

In normal ECs, PS is confined to the inner leaflet of the plasma membrane bilayer facing the cytoplasm. Exteriorization of PS is a marker for late stages of apoptosis (22). However, PS translocation to cell surfaces also accompanies the activation of platelets (23) as well as of T- and B- lymphocytes (24); in these cell types PS externalization is reversible (24,25). It would be expected that Diannexin, by binding PS on the surface of ECs and other cells during IRI, would suppress leukocyte and activated platelet attachment. This prediction is confirmed by in vivo microscopic observation of mouse liver during warm postischemic reperfusion (9). Another prediction is that Diannexin would compete with secretory phospholipase A2 (sPLA2) for binding to PS on cell surfaces. This binding is required for sPLA2 activity, which generates inflammatory mediators, such as lysophosphatidic acid (LPA) and procoagulant thromboxane (26). LPA activates platelets and binds to specific receptors on ECs, thereby inducing cytoskeletal rearrangement and cell rounding, as observed in IRI (26). An LPA3 antagonist decreases murine renal IRI (27). Diannexin reduces thromboxane production in mouse liver warm IRI (9).

These early events (attachment of leukocytes and platelets to EC and the production of pro-inflammatory mediators) trigger a cascade of events that aggravate IRI. Major inducers of adhesion molecules in ECs are TNFα and IL-1ß (28). As now reported, the expression of these molecules during liver cold IRI is decreased in recipients treated with Diannexin. Another mechanism inducing the production in ECs of ICAM-1, VCAM-1 and E-selectin, as well as chemokines, is attachment to them of activated platelets expressing CD40 ligand (28). By decreasing TNFα and IL1β expression, and preventing attachment of platelet aggregates to ECs in vivo during IRI, Diannexin would be expected to suppress the induction of integrins and chemokines. Indeed, in warm mouse liver IRI, Diannexin decreased the expression of ICAM-1, VCAM-1 and the chemokine MIP-2 (9). As now reported in rat OLTs, Diannexin markedly reduced the expression of P-selectin and IP-10, a potent chemotactic factor for activated CD4 T cells, and depressed sinusoidal endothelial cell activation, as evidenced by ICAM expression.

The consequence of these effects of Diannexin is decreased recruitment of various subsets of leukocytes into the liver during IRI, including cells with monocyte-macrophage markers, CD4+ lymphocytes and neutrophils, as now documented. Cells of monocyte-macrophage lineage are recruited in two phases of IRI: an early phase (29) and a late phase when target organ damage is already occurring. Neutrophil recruitment follows expression on ECs of major adhesion molecules such as P-selectin/ICAM-1, and contributes to the vascular inflammation, which increases in severity a few hours after the initiation of postischemic reperfusion (7). Indeed, others have shown that treatment with Annexin 1 peptides protected against ischemia-reperfusion in the heart and mesenteric microcirculation, as well as in multiple organ failure associated with splanchnic ischemia-reperfusion (30). The presently decreased CD4 T-cell infiltration in Diannexin-treated OLTs is of interest in view of evidence that this T-cell subset contributes to IRI damage (17). Supporting this viewpoint is the observation that Stat4KO mice (deficient in Th1 development) but not Stat 6KO mice were protected from the injury, whereas reconstitution of nude mice with T cells from Stat6KO, but not Stat4KO, mice restored liver IRI (31). Moreover, adoptive transfer of T cells, particularly the CD4 T subset, restored IRI in otherwise T cell-deficient and IRI-resistant hosts. Recently, we demonstrated that intra-hepatic CD4 T cells are enriched in the CXCR3+ subset, which represent pre-activated Th1 cells (32).

The inflammatory cell recruitment during IRI results in damage to ECs, including apoptosis (33). Decreased sinusoidal blood flow, in turn, results in hepatocyte death by both apoptosis and necrosis; under some dietary and other conditions necrosis predominates (34). It seems likely that early attachment of monocytes to ECs during IRI triggers apoptosis of the latter. Genetic and other studies in metazoan animals as diverse as Caenorhabditis elegans and mammals show that the binding of phagocytic cells to target cells initiates the programmed cell death occurring in normal development (35,36); this is not necessarily followed by phagocytosis (35). Tissue-targeted inactivation of macrophages prevents the apoptosis and removal of hyaloid blood vessels in the developing mouse eye (36). In an analogous manner, by suppressing the binding of monocytes to EC early in the course of IRI Diannexin would be expected to decrease EC apoptosis. As a result of improving sinusoidal blood flow during IRI, Diannexin would be expected to decrease hepatocyte apoptosis. As now reported, Diannexin significantly reduces apoptosis in OLT assayed by caspase and TUNEL positivity.

These beneficial results of Diannexin are mediated in part by modulating the expression of pro-apoptotic and anti-apoptotic genes. The protein encoded by Bcl-2 gene has been implicated in prolongation of cell survival by blocking apoptosis and preventing hepatic IRI (37). As now reported, Diannexin increased the expression of anti-apoptotic Bcl-2 and Bcl-xl. Additionally, Diannexin augments expression of anti-oxidant HO−1, thereby decreasing the production of reactive oxygen species, which promote apoptosis.

Finally, the anti-thrombotic activity of Diannexin (12) is expected to suppress microthrombus formation during liver IRI (8), further impeding blood flow, and aggravating hepatocyte damage. Remarkably, the antithrombotic effect of Diannexin is achieved with minimal effects on hemostasis (12), and no signs of increased hemorrhage. This separation of activities has important consequence in human liver transplantation. Because of decreased levels of blood coagulation proteins and platelets, these patients are prone to bleeding, and any therapy increasing the hemorrhage risk would be unacceptable. Fortunately, Diannexin appears to be safe in humans, even in concentrations greater than required to prevent IRI in OLT rat recipients.

Three questions remain to be answered. The first is whether Diannexin is efficacious when steatotic livers are transplanted. The second is whether the remarkable protection against IRI observed in rat OLT can be reproduced in human liver graft recipients. The third is whether administration of Diannexin to recipients achieves optimal protection against IRI. In this study, that regime was as efficacious as perfusion of the stored liver with Diannexin, further research following the promising lead now reported will be needed to answer these questions.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

This work was supported by NIH Grants RO1 DK062357, AI23847, AI42223 (J.W.K.W), The Roche Organ Transplantation Research Foundation (Y.Z), The Dumont Research Foundation and Alavita Inc.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References
  • 1
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