Metalloproteinase-9 deficiency protects against hepatic ischemia/reperfusion injury

Authors

  • Takashi Hamada,

    1. Dumont-UCLA Transplant Center, Division of Liver and Pancreas Transplantation, Department of Surgery, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA
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  • Constantino Fondevila,

    1. Dumont-UCLA Transplant Center, Division of Liver and Pancreas Transplantation, Department of Surgery, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA
    Current affiliation:
    1. Surgery-Transplant Liver Unit, University of Barcelona, Barcelona, Spain
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  • Ronald W. Busuttil,

    1. Dumont-UCLA Transplant Center, Division of Liver and Pancreas Transplantation, Department of Surgery, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA
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  • Ana J. Coito

    Corresponding author
    1. Dumont-UCLA Transplant Center, Division of Liver and Pancreas Transplantation, Department of Surgery, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA
    • Dumont-UCLA Transplant Center, 77-120 CHS, Box: 957054, Los Angeles, CA 90095-7054
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  • Potential conflict of interest: Nothing to report.

Abstract

Leukocyte transmigration across endothelial and extracellular matrix protein barriers is dependent on adhesion and focal matrix degradation events. In the present study we investigated the role of metalloproteinase-9 (MMP-9/gelatinase B) in liver ischemia/reperfusion (I/R) injury using MMP-9-deficient (MMP-9−/−) animals and mice treated with a specific anti-MMP-9 neutralizing antibody or with a broad gelatinase inhibitor for both MMP-9 and metalloproteinase-2 (MMP-2/gelatinase A). Compared to wild-type mice, MMP-9−/− mice and mice treated with an anti-MMP-9 antibody showed significantly reduced liver damage. In contrast, mice treated with a broad gelatinase inhibitor showed rather inferior protection against I/R injury and were characterized by persistent ongoing liver inflammation, suggesting that MMP-2 and MMP-9 may have distinct roles in this type of injury. MMP-9 was mostly detected in Ly-6G and macrophage antigen–1 leukocytes adherent to the vessel walls and infiltrating the damaged livers of wild-type mice after liver I/R injury. Leukocyte traffic and cytokine expression were markedly impaired in livers of MMP-9−/− animals and in livers of mice treated with anti-MMP-9 antibody after I/R injury; however, initiation of the endothelial adhesion cascades was similar in both MMP-9−/− and control livers. We also showed that MMP-9-specific inhibition disrupted neutrophil migration across fibronectin in transwell filters and depressed myeloperoxidase (MPO) activation in vitro. Conclusion: These results support critical functions for MMP-9 in leukocyte recruitment and activation leading to liver damage. Moreover, they provide the rationale for identifying inhibitors to specifically target MMP-9 in vivo as a potential therapeutic approach in liver I/R injury. (HEPATOLOGY 2007.)

Ischemia/reperfusion (I/R) injury is a pathophysiological process in which the hypoxic insult is further accentuated by restoration of blood flow to the compromised organ. I/R insult causes up to 10% of early transplant failures and can lead to significantly higher incidence of acute and chronic rejections.1, 2

Liver I/R damage is linked to leukocyte infiltration, release of cytokines, and free radicals.3 However, the mechanisms involved in leukocyte recruitment to inflammatory sites in liver are still not entirely understood. Overall, leukocytes circulate continuously in the blood, and their migration across endothelial barriers or extracellular matrix (ECM) proteins to inflamed tissues is a complex process dependent on the coordination of cellular adhesion-release steps and focal matrix degradation.4 Although adhesion molecules are critical to the successful promotion of leukocyte transmigration by providing leukocyte attachment to the vascular endothelium, matrix proteases are important for facilitating leukocyte movement across vascular barriers.

The matrix metalloproteinase (MMP) family comprises more than 24 well-characterized specialized zinc-dependent proteases that play key roles in the responses of cells to their microenvironment.5 Although metalloproteinase-facilitated degradation of ECM proteins is essential in physiological processes such as remodeling and tissue repair, inappropriate, prolonged, or excessive expression of these enzymes has deleterious consequences. Among the different MMPs, MMP-9, also called gelatinase B, is of particular interest.

MMP-9 is a member of the gelatinase family, which also includes MMP-2, also called gelatinase A. Gelatinases are characterized by a fibronectinlike domain of 3 type II repeats that facilitate enzyme binding to ECM substrates.6 Although MMP-2 is constitutively expressed in naive livers, MMP-9 is virtually absent; instead it is highly expressed in damaged livers after I/R injury.7 MMP-9 is produced by selected cell types, including neutrophils, monocytes/macrophages, and T lymphocytes.7–9 This inducible enzyme is responsible for the turnover and degradation of several ECM proteins, including fibronectin (FN) and type IV collagen, the major component of basement membranes.10, 11 Indeed, MMP-9 expression has been associated with several pathological conditions that require disruption of the basement membrane, such as tumor invasion,12 inflammation,11 arthritis,13 multiple sclerosis,14 systemic lupus erythematosus,15 liver I/R injury, and liver transplantation.16–18

We have previously shown that vascular expression of cellular FN, a key ECM protein expressed very early by sinusoidal endothelial cells in response to injury,19 precedes leukocyte infiltration in marginal liver I/R injury.20 We have also shown that interactions between FN and its integrin receptor α4β1 regulates the expression of MMP-9 by infiltrating leukocytes in damaged livers.7 Moreover, blockade of FN-α4β1 interactions down-regulated MMP-9 expression by leukocytes and disrupted their migration in livers after I/R injury.7 These observations supported the view that cell attachment to ECM proteins and subsequent ECM degradation are related events; they also supported the concept that MMP-9 may have a critical function in liver I/R injury. Therefore, to test whether MMP-9 is a critical factor in the cascade of events leading to liver I/R injury, our experiments included MMP-9-deficient mice, mice treated with a specific anti-MMP-9 neutralizing monoclonal antibody, and mice treated with a broad gelatinase inhibitor that targets both MMP-2 and MMP-9.

Abbreviations

ECM, extracellular matrix; FN, fibronectin; I/R, ischemia/reperfusion; ICAM-1, intercellular adhesion molecule–1; KO, knockout; Mac-1, macrophage antigen–1; MMP, metalloproteinase; MMP-2/9 inh, gelatinase inhibitor for MMP-2 and MMP-9; MMP-9−/−, MMP-9-deficient; MMP-9 mAb, anti-MMP-9 neutralizing monoclonal antibody; sGOT, serum glutamic oxaloacetic aminotransferase; sGPT, serum glutamic-pyruvic aminotransferase; VCAM-1, vascular cell adhesion molecule–1; WT, wild type.

Materials and Methods

Animals

Male MMP-9-deficient (MMP-9−/−) knockout (KO) mice (FVB.Cg-Mmp9tm1tvu; n = 16), matched MMP-9+/+ wild-type (WT) littermates (FVB/NJ; n = 16), and male C57BL6 mice (n = 40) 8 to 10 weeks of age were obtained from the Jackson Laboratory. Mice were housed in the UCLA animal facility under specific pathogen-free conditions. All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health.

Model of Partial Lobar Liver Ischemia/Reperfusion Injury

A warm hepatic I/R model was performed according to Zwacka et al.21 with minor modifications. Mice were anesthetized with sodium pentobarbital (60 mg/kg intraperitoneally) and injected with heparin (100 U/kg). Arterial and portal venous blood supplies were interrupted to the cephalad lobes of the liver for 90 minutes using an atraumatic clip. After 90 minutes of partial hepatic warm ischemia, the clip was removed, initiating hepatic reperfusion. Mice were maintained on a heating pad (37°C) to avoid temperature fall. Sham controls underwent the same surgical procedure but without vascular occlusion.

MMP-9 Targeted Therapy

C57BL6 mice were treated intravenously with an anti-MMP-9 neutralizing monoclonal antibody (clone 6-6B, Calbiochem, San Diego, CA) 3 mg/kg (MMP-9 mAb group), a broad gelatinase inhibitor (MMP-2/9 inhibitor III, H-Cys-Thr-Thr-His-Trp-Gly-Phe-Thr-Leu-Cys-OH; Calbiochem) that is a hydrophobic cyclic peptide suitable for in vivo use as an inhibitor of MMP-2 and MMP-9,22 15 mg/kg (MMP-2/9 inh group), 5 minutes prior to reperfusion. The therapeutic regimens were designed based on our observations that MMP-9 was virtually undetected in livers during interruption of the blood supply (not shown). These therapies had no effect in naive animals. Control mice were injected with isotype-matched IgG or saline in a fashion similar to that in the treated groups. An additional group of mice was treated with an anti-MMP-2 neutralizing monoclonal antibody (clone CA-4001, Fremont, CA; 3 mg/kg, i.v.) 5 minutes prior to reperfusion.

Zymography

Protein extraction and zymography analyses were performed as we have previously described.7 Briefly, gelatinolytic activity was detected in liver extracts at a final protein content of 100 μg by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) containing 1 mg/mL of gelatin (Invitrogen) under nonreducing conditions. After SDS-PAGE, the gels were washed twice in 2.5% Triton X-100 for 30 minutes each time, rinsed in water, and incubated overnight in a development buffer at 37°C [50 mmol/L Tris-HCl, 5 mmol/L CaCl2, and 0.02% NaN3 (pH 7.5)]. The gels were then stained with Coomassie brilliant blue R-250 (Bio-Rad, Hercules, CA), and destained with methanol/acetic acid/water (20:10:70). A clear zone indicated enzymatic activity. Positive controls for MMP-2 and MMP-9 (BIOMOL International, Plymouth, PA), and prestained molecular weight markers (Bio-Rad Laboratories) served as standards.

Assessment of Hepatic Function

Serum glutamic-pyruvic aminotransferase (sGPT) and serum glutamic oxaloacetic aminotransferase (sGOT) levels were measured in blood samples obtained 6 and 24 hours after hepatic reperfusion. Measurements were made with an autoanalyzer by ANTECH Diagnostics (Los Angeles, CA).

Myeloperoxidase Assasy

Myeloperoxidase (MPO) activity was evaluated as previously described.20 Frozen tissue was homogenized in an iced solution of 0.5% hexadecyltrimethyl-ammonium (Sigma, St. Louis, MO) and 50 mmol/L of potassium phosphate buffer solution (Sigma) with the pH adjusted to 5. After centrifugation, the supernatant was mixed in a solution of hydrogen peroxide–sodium acetate and tetramethyl benzidine (Sigma). The quantity of enzyme degrading in 1 μmol/L of peroxide per minute at 25°C per gram of tissue was defined as 1 U of MPO activity.

Histology and Immunohistochemistry

Liver specimens were fixed in a 10% buffered formalin solution for H&E staining or snap-frozen in liquid nitrogen for peroxidase and immunofluorescence staining, as previously described.23, 24 Appropriate primary antibodies against mouse CD4 (L3T4), macrophage antigen–1 (Mac-1; M1/70BD), Ly-6G (1A8), ICAM-1 (3E2), E-selectin (10E96), P-selectin (RB40.34), all from BD Biosciences (San Jose, CA); MMP-9 (AF909; R&D Systems, Minneapolis, MN); cellular FN (IST-9; Accurate Chemical, Westbury, NY); and vascular cell adhesion molecule–1; (VCAM-1; MVCAM A 429; Serotec Inc., Raleigh NC) were added at optimal dilutions. Bound primary Ab was detected using biotinylated antirat, biotinylated antigoat, or biotinylated antimouse IgG and streptavidin peroxidase–conjugated complexes (Vector Laboratories, Burlingame, CA). When mouse monoclonal antibodies were used, sections were blocked using a M.O.M. kit (Vector), according to the manufacturer's instructions. Negative and positive controls were included for each stain. The sections were evaluated blindly by counting the labeled cells in triplicate in 40 high-power fields per section. Dual staining was detected by immunofluorescence with Alexa Fluor 488-green antirat IgG (H+L) and Alexa Fluor 594-red antigoat IgG (H+L) antibodies (Molecular Probes, Carlsbad, CA), and slides were analyzed using a Leica Confocal Microscope (UCLA Brain Research Institute, Confocal Microscope Core Facility).

RNA Extraction and Reverse Transcriptase PCR

For evaluation of cytokine gene expression, livers were harvested, and RNA was extracted with Trizol (Life Technologies Inc., Grand Island, NY) as previously described.20 Reverse transcription was performed using 5 μg of total RNA in a first-strand cDNA synthesis reaction with SuperScript II RNaseH Reverse Transcriptase (Life Technologies), as recommended by the manufacturer. The cDNA product was amplified by PCR using primers specific for mouse cytokines and β-actin.

TNF-α Protein Expression.

Serum TNF-α protein content was determined using a commercially available ELISA kit (eBioscience, San Diego, CA) according to the manufacturer's instructions, with final results expressed in picograms of TNF-α per milliliter of serum.

Isolation of Murine Neutrophils from Bone Marrow

Isolation of adult murine neutrophils from bone marrow was performed using published methods25 with minor modifications. Briefly, femurs and tibias were harvested and stripped of all muscle and sinew, and bone marrow was flushed with 2.5 mL of RPMI-1640 containing 5% fetal bovine serum (FBS) on ice. Cells were pelleted, and erythrocytes were removed by hypotonic lysis. The entire bone marrow preparation was resuspended at 5 × 107 cells/mL in Hanks' balanced saline solution (HBSS). Cells were layered on a Percoll (Sigma–Aldrich) gradient (3 mL of 55% Percoll, top; 3 mL of 65% Percoll, middle; 4 mL of 80% Percoll, bottom) and centrifuged at 2000 rpm for 30 minutes at 10°C. Mature neutrophils were recovered at the interface of the 65% and 80% fractions and were more than 90% pure and more than 95% viable in the neutrophil-rich fraction as determined by Ly-6G immunostaining/morphology and trypan blue exclusion, respectively.

In Vitro Migration Assay

Isolated neutrophils were resuspended in RPMI-1640 without FBS at a final concentration of 2.0 × 106/mL, and transmigration through fibronectin was performed using a commercially available in vitro cell migration assay kit (BD Bioscience). Briefly, 6.5-mm-diameter Transwell inserts with 3-μm pores were used; these inserts were either coated with fibronectin or uncoated (control invasion chambers) on 24-well culture trays. Neutrophils were added at 4 × 105 cells/well to the top chamber with or without different doses (0, 1, 10, and 50 nM) of MMP-9 inhibitor-I (C27H33N3O5S; Calbiochem), which is suitable for specifically inhibiting MMP-9 in vitro,26 and the chemoattractant formyl-Met-Leu-Phe-OH (fMLP; 10 nM; Calbiochem) was added to the lower chamber. The neutrophils were incubated at 37°C and 5% CO2 for 4 hours, and the cells that had migrated into the lower chamber were collected, stained with Mayer's Hematoxylin solution (Sigma), and counted.

In Vitro Myeloperoxidase Activation Assay

Isolated neutrophils were resuspended in FBS-free RPMI-1640 medium at a final concentration of 1.0 × 106/mL. Neutrophils were activated with fMLP (10 nM) and incubated at 37°C in 5% CO2 for 2 hours with or without MMP-9 inhibitor I (Calbiochem) at a final concentration of 0, 1, 10, or 50 nM. MPO activity was then measured in the cell supernatants according to the standard protocol and expressed as change in absorbance per minute per milliliter of supernatant.

Statistical Analysis

Results are expressed as mean ± standard deviation. Statistical comparisons between groups of normally distributed data were performed with the Student t test using the statistical package SPSS (SPSS Inc., Chicago, IL). P values less than 0.05 were considered statistically significant.

Results

Characterization of MMP-9 Deficiency.

Zymography analysis showed no MMP-9 activity in naive MMP-9−/− (KO) livers and naive MMP-9+/+ (WT) livers. However, MMP-9 activity was highly expressed in the MMP-9+/+ wild-type control livers 6 and 24 hours after liver I/R injury and was indeed undetectable in the corresponding MMP-9-deficient livers (Fig. 1). These results were consistent with our previous observations in a rat model of steatotic liver transplantation, in which MMP-9 was highly expressed after liver I/R injury.7 MMP-2 activity was mildly detected in naive MMP-9−/− and wild-type livers and was up-regulated in MMP-9−/− (KO) livers and matched wild-type livers after I/R insult (Fig. 1).

Figure 1.

Representative gelatin zymography in MMP-9−/− and MMP-9+/+ mice. MMP-9 activity was highly expressed in MMP-9+/+ (WT) livers 6 hours (lanes 1–3) and 24 hours (lane 7) after reperfusion following 90 minutes of warm ischemia. MMP-9−/− (KO) livers showed no MMP-9 activity 6 hours (lanes 4–6) and 24 hours (lane 8) after I/R injury. MMP-9 activity was virtually undetectable in naive livers (lane 9). MMP-2 activity was detected in naive livers and in MMP-9+/+ (WT) and MMP-9−/− (KO) livers after liver I/R injury.

MMP-9 Deficiency Improved Liver Function and Histology after I/R Injury.

Our earlier studies in a model of steatotic liver transplantation provided evidence that MMP-9 is a player in liver I/R injury. To examine whether a lack of MMP-9 activity would confer protection against hepatic I/R injury, we used MMP-9-deficient mice in a well-established model of liver I/R injury. MMP-9−/− mice showed significantly less liver damage, as shown by the reduced aminotransferase levels (sGPT: 1215 ± 463 versus 5482 ± 931 U/L, P < 0.0003; sGOT: 5706 ± 1940 versus 15,790 ± 2090 U/L, P < 0.0001; n = 6–8/group) 6 hours after I/R injury (Fig. 2A). A sustained effect was observed in the MMP-9−/− mice, with levels of sGPT (726 ± 881 versus 7172 ± 4829 U/L; P < 0.03, n = 6/group) and sGOT (727 ± 421 versus 4248 ± 2891 U/L; P < 0.03; n = 6/group) depressed 24 hours after I/R injury (Fig. 2A,B). Moreover, decreased liver aminotransferase level in the MMP-9−/− mice was associated with significantly better histological preservation (Fig. 2C).

Figure 2.

Liver aminotransferase and histological preservation in MMP-9−/− and MMP-9+/+ mice. (A) sGPT and (B) sGOT levels (IU/L) were measured in blood samples taken 6 and 24 hours after I/R injury. sGPT and sGOT levels in the MMP-9−/− mice were significantly lower than those in their respective MMP-9+/+ control littermates after 6 hours (*P < 0.0003, §P < 0.0001; n = 6–8/group) and after 24 hours (**P < 0.03; n = 6/group). (C) Representative H&E staining of livers 6 and 24 hours post-I/R injury. Control MMP-9+/+ livers were characterized by elevated sinusoidal congestion after 6 hours (a) and extensive necrosis after 24 hours (b). In contrast, after 6 hours (c) and 27 hours (d) MMP-9−/− livers showed almost no vascular congestion and sparse focal necrotic areas (original magnification: ×100).

Elevated sinusoidal congestion and extensive areas of necrosis characterized livers from wild-type mice 6 and 24 hours post-I/R injury, respectively. In contrast, MMP-9-KO mice showed only mild signs of vascular change and necrosis after liver I/R injury.

Anti-MMP-9 Antibody Therapy Was Highly Effective in Protecting against Liver I/R Injury.

Knockout mice are an important research tool; however, they often have redundant mechanisms. Therefore, additional experiments were performed in wild-type C57BL6 mice treated with a neutralizing monoclonal antibody against MMP-9 (MMP-9 mAb) or with a broad gelatinase inhibitor for MMP-2 and MMP-9 (MMP-2/9 inh) prior to reperfusion. Mice in which MMP-9 was specifically targeted with the MMP-9 mAb showed a marked decrease in liver damage 6 hours after I/R injury (sGPT: 2443 ± 809 versus 5485 ± 582 U/L; P < 0.003; sGOT: 1176 ± 324 versus 19,516 ± 5812 IU/L; P < 0.002; n = 5/group) compared with that in their respective IgG-treated controls (Fig. 3A,B). Furthermore, livers treated with MMP-9 mAb showed excellent histological preservation, whereas control livers were characterized by elevated sinusoidal congestion and focal necrosis (Fig. 3C).

Figure 3.

Liver aminotransferases and histological preservation in MMP-9 mAb– and MMP-2/9 inh–treated mice. (A) sGPT and (B) sGOT levels (IU/L) measured in blood samples taken 6 hours after I/R injury. sGPT and sGOT levels in the MMP-9 mAb mice were significantly lower than those in their respective controls (*P < 0.003, §P < 0.002; n = 5/group). Livers treated with MMP-2/9 inh showed only a slight improvement in liver enzyme levels (**P < 0.05; n = 5/group). (C) Representative H&E staining of livers 6 hours post-I/R injury. MMP-9 mAb–treated livers were characterized by very good histological preservation (c), contrasting with their respective control livers, which showed elevated vascular congestion and signs of necrosis (a). Livers treated with MMP-2/9 inh (d) and their respective controls (b) both characterized by elevated vascular congestion. (Original magnification ×100).

We also evaluated the efficacy of a broad gelatinase inhibitor in the mouse model of liver I/R injury (no specific MMP-9 inhibitors are available for in vivo use). Surprisingly, mice treated with the MMP-2/9 inh showed only a modest decrease in aminotransferase level (sGPT: 4100 ± 655 versus 4933 ± 378 U/L; sGOT: 11,794 ± 1760 versus 16,071 ± 3732 U/L; P < 0.05; n = 5/group), and the livers of both the MMP-2/9 inh–treated and control mice showed significant signs of vascular congestion (Fig. 3). Moreover, aminotransferase levels were comparable in the MMP-2/9 inh–treated group and the matching wild-type controls 24 hours after reperfusion (not shown). To evaluate whether inhibition of MMP-2 would be detrimental in our model, additional mice were treated with an anti-MMP-2 neutralizing monoclonal antibody (MMP-2 mAb). MMP-2 mAb–treated mice showed about a 7-fold increase in sGPT (43,925 ± 13,169 versus 6072 ± 952 U/L, P < 0.006; n = 4/group) and an sGOT level (16,200 ± 6789 versus 14,322 ± 4838 U/L; n = 4/group) comparable to that in the IgG-treated controls 6 hours after liver I/R injury. All together, our observations indicate that specific MMP-9 inhibition was important for protecting against liver I/R injury.

Blocking MMP-9 Decreased Myeloperoxidase Activity in Liver I/R Injury.

Myeloperoxidase, one of the most abundant proteins in neutrophils, representing 5% of total protein,27 has emerged as an enzyme critically involved in the pathogenesis of inflammatory diseases.28 In our liver I/R model, MPO activity (U/g) 6 hours after reperfusion was significantly depressed in MMP-9−/− mice (3.5 ± 1.5 versus 10.7 ± 2.1 U/g, P < 0.002), in mice treated with MMP-9 mAb (3.1 ± 1.7 versus 13.72 ± 3.3 U/g, P < 0.001), and in mice treated with MMP-2/9 inh (6.9 ± 0.8 versus 15.4 ± 3.3 U/g, P < 0.05), compared with that in their respective controls (Fig. 4). Therefore, these results provide evidence of a strong correlation between MMP-9 inhibition and reduced MPO activation in liver I/R injury.

Figure 4.

Intrahepatic MPO enzyme activity (units per gram) in MMP-9−/− mice, mice treated with MMP-9 mAb, and mice treated with MMP-2/9 inh. MPO enzymatic activity, determined 6 hours after reperfusion following 90 minutes of warm ischemia, was significantly reduced in the MMP-9−/− (*P < 0.0002; n = 6/group), MMP-9 mAb (**P < 0.001; n = 5/group), and MMP-2/9 inh (***P < 0.006; n = 5/group) groups compared with that in their respective controls. In addition, MMP-9−/− livers also showed markedly lower MPO activity 24 hours post-I/R injury (****P < 0.0001; n = 6/group).

Blocking MMP-9 Impaired Leukocyte Accumulation and Activation in Liver I/R Injury.

Leukocyte migration across endothelial or ECM barriers is dependent on focal matrix degradation.29 MMP-9 staining, absent in naive livers, was predominantly detected in infiltrating Ly-6G and Mac-1 leukocytes after liver I/R injury, as clearly shown by double immunofluorescence and confocal microscopy analysis (Fig. 5). Leukocytes were negative for MMP-9 before initiating the adhesion/transmigration process and expressed MMP-9 during recruitment to livers after I/R injury (Fig. 5). Ly-6G is expressed primarily in granulocytes,30 and Mac-1 is a mouse macrophage antigen abundantly expressed in stimulated macrophages and, to a lesser degree, in granulocytes.31

Figure 5.

Colocalization of MMP-9 and leukocyte markers in livers after I/R injury. All images are confocal optical sections of immunostained WT livers 6 hours post-I/R injury. (A) Ly-6G cells stained in green (Alexa Fluor 488). (B) MMP-9 labeled in red (Alexa Fluor 594). (C) Cell colocalization of Ly-6G/MMP-9 marker shown in yellow-orange. (D) Mac-1 cells stained in green (Alexa Fluor 488). (E) MMP-9 labeled in red (Alexa Fluor 594). (F) Cell colocalization of Mac-1/MMP-9 marker shown in yellow-orange. Open arrow, green leukocyte: leukocytes negative for MMP-9 before initiating the adhesion/transmigration process; filled arrow, yellow leukocyte: leukocytes expressing MMP-9 during transmigration.

Next, we determined whether targeting MMP-9 affected leukocyte infiltration in liver I/R injury. MMP-9−/− livers showed significantly reduced numbers of CD4 T cells (16 ± 3 versus 29 ± 5, P < 0.01), Mac-1 cells (44 ± 9 versus 62 ± 11, P < 0.05), Ly-6G neutrophils (46 ± 5 versus 92 ± 7, P < 0.05), and MMP-9 cells (0 versus 94 ± 5; P < 0.0001) in periportal areas 6 hours after liver I/R injury (Figs. 6 and 7A). Leukocyte infiltration was also clearly depressed in the periportal areas of MMP-9 mAb–treated livers (CD4: 14 ± 2 versus 21 ± 7, P < 0.05; Mac-1: 63 ± 4 versus 86 ± 5, P < 0.01; Ly-6G: 46 ± 4 versus 94 ± 9, P < 0.03; and MMP-9: 39 ± 3 versus 94 ± 9, P < 0.01; Fig. 7B). In contrast, the MMP-2/9 inh–treated group showed reduced numbers of CD4 cells (13 ± 2 versus 21 ± 6, P < 0.01) and MMP-9 cells (72 ± 6 versus 94 ± 9, P < 0.05), but almost no change in the numbers of Mac-1 (83 ± 4 versus 86 ± 5) and Ly-6G (82 ± 5 versus 94 ± 9) leukocytes (Fig. 7B). The extent of leukocyte infiltration and positioning in portal areas was correlated with the degree of liver damage observed in the different groups; it was also correlated to some extent with the expression of proinflammatory cytokines. Indeed, proinflammatory cytokines were significantly depressed in MMP-9−/− livers (TNF-α, P < 0.05; IL-6, P < 0.05; IL-2, P < 0.01; IFN-γ, P < 0.05) and in MMP-9 mAb livers (TNF-α, P < 0.05; IL-2, P < 0.01; IFN-γ, P < 0.05), shown in Fig. 8. Moreover, serum TNF-α level was markedly reduced in MMP-9−/− (P < 0.002) and MMP-9 mAb–treated (P < 0.004) mice (Fig. 9). However, cytokine expression in the MMP-2/9 inh-treated group was comparable to that in the controls (Figs. 8 and 9), suggesting that to some extent MMP-2 inhibition may overcome the beneficial function of MMP-9 inhibition in this experimental group.

Figure 6.

Immunostaining of leukocytes in peiportal areas. (A) Staining of CD4 lymphocytes in MMP-9+/+ WT livers 6 hours post-I/R injury. (B) Staining of Mac-1 cells in MMP-9+/+ WT livers 6 hours post-I/R injury. (C) Staining of Ly-6G neutrophils in MMP-9+/+ WT livers 6 hours post-I/R injury. (D) Staining of MMP-9 cells in MMP-9+/+ WT livers 6 hours post-I/R injury. (E) Staining of CD4 lymphocytes in MMP-9−/− livers 6 hours post-I/R injury. (F) Staining of Mac-1 cells in MMP-9−/− livers 6 hours post-I/R injury. (G) Staining of Ly-6G neutrophils in MMP-9−/− livers 6 hours post-I/R injury. (H) Staining of MMP-9 cells in MMP-9−/− livers 6 hours post-I/R injury 6 hours post-I/R injury. There were significantly fewer CD4, Mac-1, and Ly-6G leukocytes in the periportal areas of MMP-9-deficient livers compared with the levels in their respective controls. MMP-9 staining was negative in the MMP-9−/− livers, whereas MMP-9-positive leukocytes were found to adhere to the vessel walls and to have infiltrated the wild-type livers (arrows). Original magnification: ×200.

Figure 7.

Leukocyte infiltration in MMP-9−/− livers, livers treated with MMP-9 mAb, and livers treated with MMP-9 inh. (A) Sequential immunostaining analysis of infiltrating cells in MMP-9−/− mice 6 hours post-I/R injury. (B) Sequential immunostaining analysis of infiltrating cells in mice treated with a neutralizing antibody, anti-MMP-9 (MMP-9 mAb), or a broad gelatinase inhibitor (MMP-2/9 inh) 6 hours post-I/R injury. The results of immunostaining are expressed as the mean ± SD of data from 40 high-power fields per section per mouse and 5–6 mice per group (§P < 0.0001, *P < 0.01, &P < 0.03, **P < 0.05).

Figure 8.

Proinflammatory cytokine gene expression in MMP-2/9−/− livers, livers treated with MMP-9 mAb, and livers treated with MMP-9 inh. Cytokine induction ratios were determined 6 hours after reperfusion following 90 minutes of warm ischemia. Proinflammatory TNF-α, IL-6, IL-2, and IFN-γ expression was profoundly depressed in MMP-9-deficient livers compared with that in their respective controls. MMP-9 mAb–treated livers were characterized by significantly decreased levels of TNF-α, IL-2, and IFN-γ, whereas MMP-2/9 inh–treated livers did not significantly differ in expression of proinflammatory cytokines. Means ± SDs of 5–6 experiments are shown (*P < 0.05, **P < 0.01).

Figure 9.

Serum TNF-α levels in MMP-2/9−/− livers, livers treated with MMP-9 mAb, and livers treated with MMP-9 inh. Serum TNF-α level was profoundly depressed in MMP-9-deficient (*P < 0.002), and in MMP-9 mAb–treated (**P < 0.004) mice 6 hours after liver I/R injury. MMP-2/9 inh–treated mice showed no significant differences with the controls in TNF-α level.

Deficiency in MMP-9 Did Not Disturb Initial Expression of Vascular Adhesion Molecules in Liver I/R Injury.

To evaluate whether the observed decrease in leukocyte migration was associated with possible modifications of the expression of vascular adhesion molecules in the MMP-9-deficient livers, we assessed the expression of major adhesion molecules after liver I/R injury. Intercellular adhesion molecule–1 (ICAM-1) was modestly expressed in naive livers, whereas cellular FN and VCAM-1 were virtually absent from these livers (Fig. 10). In contrast, all 3 adhesion molecules were readily detected in livers 6 hours after I/R injury in both MMP-9−/− and MMP-9+/+ WT mice (Fig. 10). Although portal and sinusoidal endothelia were positive for cellular FN and ICAM-1 staining, VCAM-1 labeling was mostly restricted to some portal tract vessels. Cellular FN, ICAM-1, and VCAM-1 showed comparable patterns of expression in livers from both MMP-9−/− and MMP-9+/+ WT mice 6 hours after I/R injury (Fig. 10). E-selectin and P-selectin were mildly and almost not expressed in both MMP-9−/− and wild-type livers 6 hours after I/R injury (not shown), which is consistent with the observations that selectins have a minimal role in recruitment of leukocytes to inflamed liver microvasculature.32 Therefore, our results indicate that the initial adhesion steps were equally initiated in both MMP-9−/− and wild-type livers after I/R injury and provide evidence that disruption of cell migration was caused by focal matrix degradation mechanisms.

Figure 10.

Expression of vascular adhesion molecules in MMP-9−/− livers. (A) Sequential immunostaining analysis of cellular FN in naive livers 6 hours post-I/R injury. (B) Sequential immunostaining analysis of cellular FN in MMP-9+/+ WT mice 6 hours post-I/R injury. (C) Sequential immunostaining analysis of cellular FN in MMP-9−/− littermates 6 hours post-I/R injury. (D) Sequential immunostaining analysis of VCAM-1 in naive livers 6 hours post-I/R injury. (E) Sequential immunostaining analysis of VCAM-1 in MMP-9+/+ WT mice 6 hours post-I/R injury. (F) Sequential immunostaining analysis of VCAM-1 in MMP-9−/− littermates 6 hours post-I/R injury. (G) Sequential immunostaining analysis of ICAM-1 in naive livers 6 hours post-I/R injury. (H) Sequential immunostaining analysis of ICAM-1 in MMP-9+/+ WT mice 6 hours post-I/R injury. (I) Sequential immunostaining analysis of ICAM-1 in MMP-9−/− littermates 6 hours post-I/R injury. Cellular FN and VCAM-1 expression was virtually negative, whereas ICAM-1 was readily detected in naive livers. Six hours after liver I/R injury, cellular FN and ICAM-1 were detected in the portal and sinusoidal endothelia, whereas VCAM-1 was mostly restricted to the portal tract endothelia of both MMP-9−/− livers and their respective controls.

MMP-9 Inhibition Impaired Neutrophil Migration Across Fibronectin.

Neutrophils are considered critical mediators in acute inflammatory liver injury.33 Our in vivo studies showed that specific inhibition of MMP-9 significantly decreased neutrophil positioning in the portal areas. The recruitment of leukocytes toward inflammatory sites follows a sequential multistep process; although adhesion molecules are critical to promoting adhesion to the vascular endothelium and initiate transmigration, they may also act as additional barriers that leukocytes have to cross during migration. We have recently shown that leukocyte–FN interactions up-regulated MMP-9 expression in damaged livers.7 Cellular FN, which was absent from naive livers, was expressed in both portal and sinusoidal endothelia 6 hours after I/R injury in both MMP-9-deficient and WT mice (Fig. 10). To test whether MMP-9 inhibition had an effect on neutrophil migration in fibronectin, we performed a series of migration assays in the presence of several concentrations of MMP-9 inhibitor-I, a specific MMP-9 inhibitor suitable for in vitro use.26 We observed that the presence of MMP-9 inhibitor-I at all the studied doses did not disrupt neutrophil migration in the absence of an ECM barrier, as neutrophils passed unimpeded through the 3-μm pores of the control invasion chambers in the same numbers. However, when neutrophil migration was performed across FN, cell migration was delayed, becoming further reduced when neutrophils were treated with an MMP-9 inhibitor. As Fig. 11 shows, MMP-9-specific inhibition significantly decreased neutrophil migration across FN in a dose-dependent manner.

Figure 11.

Neutrophil migration across fibronectin. Migration of neutrophils across fibronectin was impaired in the presence of MMP-9 inhibitor-I in a dose-dependent manner. Percentages of cell migration after 4 hours of incubation are expressed as the mean ± SD of 3 experiments. The paired Student t test was used to compare transmigration across fibronectin in the absence of MMP-9 inhibitor-I with migration across fibronectin in the presence of different doses of MMP-9 inhibitor-I (§P < 0.003, *P < 0.002, **P < 0.004).

MMP-9 Inhibition Impaired Myeloperoxidase Activation In Vitro.

Specific inhibition of MMP-9 was very effective in depressing neutrophil migration, both in vivo and in vitro. However, our in vivo observations indicated that neutrophil migration and MPO activation were affected at different levels after I/R injury, raising the possibility that in addition to cell migration, MMP-9 inhibition may also affect neutrophil-derived MPO activity. For instance, in the MMP-9 mAb group, neutrophil migration showed about a 2-fold decrease, whereas MPO activity showed about a 3-fold reduction after liver I/R injury (Figs. 4 and 7). In addition, neutrophil infiltration was only slightly changed in the MMP-2/9 inh–treated livers, contrasting with MPO activity, which was significantly decreased in these livers (Figs. 4 and 7). To verify whether MMP-9 inhibition was also able to regulate MPO activity, we cultured fMLP-stimulated neutrophils in the presence of different concentrations of MMP-9 inhibitor-I. As illustrated in Fig. 12, MMP-9 inhibition did not affect MPO activity in nonstimulated neutrophils. MPO activity was increased in fMLP-stimulated neutrophils; however, in the presence of MMP-9 inhibitor-I, fMLP-stimulated neutrophils showed reduced MPO activity. This effect was highly manifest at an MMP-9 inhibitor-I concentration of 50 nM (P < 0.05). All together our results provide an indication that MMP-9 inhibition regulates both neutrophil transmigration and MPO activation.

Figure 12.

Inhibition of myeloperoxidase activation in vitro. MPO activity in nonstimulated neutrophils (open bar) was very low and was unchanged by the presence of 50 nM of MMP-9 inhibitor-I (hatched bar). FMLP-stimulated neutrophils (solid bars) showed high MPO activity, which was reduced by MMP-9 inhibitor-I. MPO activity was profoundly reduced at an inhibitor concentration of 50 nM and was comparable to that measured in nonstimulated neutrophils. Data are shown as mean ± SD (n = 3, *P < 0.05).

Discussion

In the present study, we investigated the functional significance of MMP-9 in liver I/R injury in a well-established mouse model of warm liver I/R injury. Our major findings were: (1) MMP-9-deficient mice and mice treated with anti-MMP-9 neutralizing monoclonal antibodies showed profound improvement in serum aminotransferases and liver histological outcomes; (2) in contrast, mice treated with a broad gelatinase inhibitor that simultaneously inhibits MMP-2 and MMP-9 exhibited rather modest protection from liver injury; (3) Mac-1 and Ly-6G leukocytes were major sources of MMP-9 in damaged wild-type mouse livers; (4) MMP-9 deficiency impaired leukocyte recruitment; (5) this impairment occurred without disturbing the initial expression of major vascular adhesion molecules, such as cellular FN, VCAM-1, and ICAM-1; (6) MMP-9 deficiency and anti-MMP-9 antibody therapy depressed the expression of TNF-α, IL-2, and IFN-γ, whereas broad gelatinase inhibition was inefficient in depressing proinflammatory cytokine expression; (7) the in vitro investigation provided evidence that MMP-9 was an active player in neutrophil migration across fibronectin; and (8) the in vitro investigation also showed that MMP-9 actively assisted myeloperoxidase activation.

Liver I/R injury, a multifactorial antigen-independent inflammatory process, leads to a decline in liver function and to a potential increase in organ immunogenicity, which can result in graft loss. MMP-9 has been implicated as an important enzyme during inflammation because of its ability to facilitate leukocyte trafficking through the ECM.34 We have previously shown that MMP-9 expression in damaged livers is part of a cascade of events occurring during I/R injury.7 Others have established a correlation between disease severity/progression and MMP-9 detection in the serum of patients with various types of liver injury, including I/R injury,16 acute allograft rejection,18 and chronic viral hepatitis.35 In the present study, we have shown that MMP-9 deficiency significantly depressed sGPT and sGOT levels and improved liver histology, providing an indication that MMP-9 is indeed an active player in liver I/R injury. Moreover, wild-type mice treated with a specific anti-MMP-9 neutralizing antibody before reperfusion also showed a marked decrease in liver damage.

Although MMP-9−/−-deficient mice and mice treated with the anti-MMP-9 antibody were found to be significantly protected against liver I/R injury, treatment of mice with a broad gelatinase inhibitor, which inhibits both MMP-2 and MMP-9, was found to have very little effect on aminotransferase and histological outcomes, providing an indication that the 2 MMPs may play opposite roles in liver I/R injury. Indeed, mice treated with an anti-MMP-2 neutralizing antibody were characterized by intensified liver damage after I/R injury when compared with the already severely injured controls. The different roles the 2 gelatinases may have are possibly caused by differences in cellular sources and enzymatic substrates. MMP-9 is an inducible gelatinase expressed mostly by leukocytes, whereas MMP-2 is generally constitutively expressed and is derived largely from stromal cells and not usually expressed by leukocytes.36 Indeed, MMP-9 is mostly detected in infiltrating leukocytes in liver I/R injury.7 In our mouse model of warm liver I/R injury, both Ly-6G neutrophils and macrophage-1 antigen–positive (Mac-1) leukocytes were major sources of MMP-9 in the damaged wild-type livers. Moreover, leukocytes expressed MMP-9 during adhesion to the vessel wall and during transmigration; leukocytes were virtually negative for MMP-9 before initiation of the adhesion/transmigration process. We have also previously shown that macrophages cultured on fibronectin express high levels of MMP-9 and virtually no MMP-2.7 Interestingly, it has been demonstrated that MMP-2 is able to cleave monocyte chemoattractant protein–3 (MCP-3) and that the cleaved molecule acts as a general chemokine antagonist, depressing inflammation, whereas MMP-9 is not capable of cleaving MCP-3.37 Interestingly, MMP-2-deficient mice exhibit severe autoimmune encephalomyelitis because of an increase in MMP-9 activity,38 suggesting that inhibition of MMP-2 may result in stimulation of MMP-9. Furthermore, an elegant study using a mouse model of rheumatoid arthritis showed that MMP-9−/− and MMP-2−/− mice have reduced and exacerbated arthritis, respectively, compared with wild-type controls; however, double MMP-2/MMP-9-deficient mice showed no significant differences with the wild-type controls in the extent of arthritis.39 All together, these observations support the view that MMP-2 expression, in contrast with expression of MMP-9, may play a regulatory role in the cessation of inflammation in liver I/R injury. Further experimentation is required to test this hypothesis.

Matrix degradation is essential to leukocyte migration across ECM proteins, not only by facilitating “matrix permeability,” but also by generating ECM-derived fragments, which are biologically active and can be highly chemotactic for leukocytes.40 MMP-9 deficiency and specific anti-MMP-9 antibody therapy clearly depressed the infiltration of CD4, Ly-6G, and Mac-1 leukocytes in periportal areas after I/R injury. On the other hand, a broad gelatinase inhibitor only had a very limited effect on the number of leukocytes detected in the liver after I/R injury, in particular the numbers of Ly-6G and Mac-1 cells. Previously, it was reported that MMP-9 promotes T-cell and neutrophil recruitment in liver I/R injury.41 Although this study supports the potential of MMP-9-mediated leukocyte recruitment, it did not adequately evaluate the function of MMP-9, because the authors used in their studies a gelatinase inhibitor for both MMP-2 and MMP-9 and evaluated liver specimens after fewer than 3 hours of reperfusion. In general, leukocyte infiltration is rather modest before 3 hours of liver I/R injury. Our observations are in line with murine models of allergen-induced airway inflammation and zymosan peritonitis, in which MMP-9-deficient mice showed significantly less leukocyte infiltration compared with that in their corresponding wild-type littermates.42, 43

Leukocyte migration was impaired in the MMP-9-deficient mice independently of the up-regulation of major vascular cell adhesion molecules. Indeed, the early expression of cellular FN, VCAM-1, and ICAM-1 was similar in both MMP-9-deficient and wild-type livers after I/R. FN and ICAM-1 were up-regulated in the portal and sinusoidal endothelia, whereas VCAM-1 expression was mostly restricted to the portal tract vessels. Neutrophils are critical mediators of liver injury, and their migration into the liver parenchyma is required for neutrophil-induced liver injury.33 Interestingly, in vitro neutrophil migration across fibronectin was significantly disrupted by the presence of a specific inhibitor of MMP-9. Fibronectin is a substrate for MMP-9 that has been shown to be expressed by sinusoidal endothelial cells very early after injury19 and to precede leukocyte infiltration in damaged livers.7 Overall, these observations indicate that the initial adhesion steps were equally initiated in both MMP-9−/− and wild-type livers after I/R injury and provide evidence that the disruption in cell traffic observed in the MMP-9-deficient mice was caused by impairment of the focal matrix degradation mechanisms.

In addition to showing leukocyte migration, MMP-9-deficient mice and mice treated with anti-MMP-9 antibody were characterized by a profound reduction in the expression of proinflammatory cytokines after liver I/R injury. In contrast, mice treated with a broad gelatinase inhibitor showed elevated expression of TNF-α and other proinflammatory cytokines, indicating that inflammation in these animals was still an ongoing process. TNF-α, which is a potent proinflammatory cytokine, is often associated with neutrophil infiltration and liver damage.

Myeloperoxidase, a heme protein abundantly expressed in neutrophils and accounting for up to 5% of total cell protein,44 is likely an important factor in neutrophil-induced liver injury. MPO generates cytotoxic oxidants such as nitric oxide (NO)–derived inflammatory oxidants.45 The MPO-dependent pathways for formation of reactive nitrogen species may be particularly relevant in liver I/R injury because after I/R damage livers express high levels of iNOS,20 which generates large amounts of NO.46 In our experimental settings, MPO activation was profoundly depressed after I/R in the MMP-9-deficient mice, in the mice treated with the neutralizing anti-MMP-9 antibody, and in the mice treated with the broad gelatinase inhibitor, suggesting that in addition to mediating leukocyte recruitment, MMP-9 may also facilitate MPO activation. These observations were supported by the cell culture assays, in which activated neutrophils showed lower MPO activity in the presence of a specific MMP-9 inhibitor. The mechanism of MPO activation by MMP-9 may be similar to the mechanisms involved in the activation of TNF-α or α-defensin, in which the precursors are cleaved to biologically active mature forms by metalloproteinases.47, 48

Although our work strongly supports the concept that MMP-9 inhibition is important in liver I/R injury and likely in several other inflammatory diseases, it should be taken into consideration that inhibition of MMP-949 or other proinflammatory mediators like TNF-α50 may be unfavorable in conditions like liver regeneration after hepatectomy. In a situation in which liver mass has to be substantially restored, liver cells need to acquire functions such as cell migration, and some of these functions may be facilitated by MMP-9.

In summary, the objective of this study was to investigate the role of MMP-9 in liver I/R injury. This study is the first to show that specifically targeting MMP-9 profoundly ameliorated tissue damage after warm liver I/R insult. Compared with wild-type mice, MMP-9-deficient mice and mice treated with a specific neutralizing anti-MMP-9 antibody showed significantly better liver preservation outcomes. This study also provides evidence that specifically targeting MMP-9 leads to more effective protection against liver I/R injury than simply using a broad gelatinase inhibitor. Overall, MMP-9 expressed by leukocytes is likely to be a key factor in cell transmigration and activation leading to liver injury. Thus, this work strongly supports the rationale for identifying inhibitors that specifically target MMP-9 in vivo as a potential therapeutic approach in the pathogenesis of liver I/R injury.

Acknowledgements

We thank Sergio Duarte for his help with the confocal microscopy pictures.

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