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Originally published online as doi:10.1189/jlb.0805468 on March 21, 2006

Published online before print March 21, 2006
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(Journal of Leukocyte Biology. 2006;79:1295-1305.)
© 2006 by Society for Leukocyte Biology

Matrix metalloproteinase-9 promotes neutrophil and T cell recruitment and migration in the postischemic liver

Andrej Khandoga*,1, Julia S. Kessler*, Marc Hanschen*, Alexander G. Khandoga*, Dorothe Burggraf{dagger}, Christoph Reichel*, Gerhard F. Hamann{dagger}, Georg Enders* and Fritz Krombach*

* Institute for Surgical Research and
{dagger} Department of Neurology, University of Munich, Germany

1Correspondence: Institute for Surgical Research, University of Munich, Marchioninistr. 27, D-81377 Munich, Germany. E-mail: andrej.khandoga{at}med.uni-muenchen.de


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ABSTRACT
 
Matrix metalloproteinases-2 and -9 (MMP-2/9) are critically involved in degradation of extracellular matrix, and their inhibition is discussed as a promising strategy against hepatic ischemia-reperfusion (I/R) injury. Here, we analyzed the role of MMP-2 and -9 for leukocyte migration and tissue injury in sham-operated mice and in mice after I/R, treated with a MMP-2/9 inhibitor or vehicle. Using zymography, we show that the MMP-2/9 inhibitor abolished I/R-induced MMP-9 activation, whereas MMP-2 activity was not detectable in all groups. As demonstrated by intravital microscopy, MMP-9 inhibition attenuated postischemic rolling and adherence of total leukocytes in hepatic postsinusoidal venules, CD4+ T cell accumulation in sinusoids, and neutrophil transmigration. These effects were associated with reduction of plasma tumor necrosis factor {alpha} (TNF-{alpha}) levels and endothelial expression of CD62P. Motility of interstitially migrating leukocytes was assessed by near-infrared reflected light oblique transillumination microscopy in the postischemic cremaster muscle. Upon MMP-9 blockade, leukocyte migration velocity and curve-line and straight-line migration distances were reduced significantly as compared with the vehicle-treated I/R group. Postischemic sinusoidal perfusion failure, hepatocellular apoptosis, and alanine aminotransferase activity were only slightly reduced after MMP-9 inhibition, whereas aspartate aminotransferase activity and mortality were significantly lower. In conclusion, MMP-9 is involved in the early recruitment cascades of neutrophils and CD4+ T cells, promotes neutrophil and T cell transmigration during hepatic I/R, and is required for motility of interstitially migrating leukocytes. MMP-9 blockade is associated with an attenuation of TNF-{alpha} release and endothelial CD62P expression, weakly protects from early microvascular/hepatocellular I/R damage, but improves postischemic survival.

Key Words: ischemia-reperfusion • leukocyte transmigration • leukocyte motility • CD4+ T cells • microvascular injury • intravital microscopy


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INTRODUCTION
 
Ischemia/reperfusion (I/R) injury remains the main reason for hepatic dysfunction after liver transplantation and major resection. Inhibition of matrix metalloproteinases (MMPs) is discussed as an appealing target and a promising strategy for pharmacological interventions against postischemic hepatic damage [1 ]. A potential clinical relevance of MMP blockade is outlined by recent reports in which nonselective inhibition of MMPs protects from hepatic I/R injury [2 3 4 ]. MMPs include a group of more than 20 zinc-dependent endopeptidases expressed in endothelial and Kupffer cells as well as in different subsets of leukocytes. MMP-2 (gelatinase-A) and -9 (gelatinase B) seem to be of particular relevance to the liver, as they are critically involved in the degradation of components of the basement membrane such as collagen IV and fibronectin, two main components of the space of Disse.

MMPs do not only regulate the release of proinflammatory cytokines {tumor necrosis factor {alpha} (TNF-{alpha}), interleukin (IL)-1ß [5 , 6 ]} and the expression of adhesion molecules (CD31, ß4-integrin, E-cadherin [7 8 9 ]) but may also facilitate leukocyte extravasation by destroying components of the extracellular matrix (ECM) during an inflammatory response. Therefore, it appears likely that gelatinases MMP-2 and -9 are involved in the process of leukocyte activation, leukocyte transmigration across the endothelial layer, as well as leukocyte migration in the hepatic parenchyma to the afflicted sites during postischemic inflammatory reaction. The question of whether MMP-2/9 are involved in the cascades of leukocyte migration in the postischemic liver has not yet been investigated. Therefore, the aim of this in vivo study was to investigate the role of MMP-2 and -9 for migration of leukocytes, in particular, neutrophils and CD4+ T cells, during hepatic I/R and to analyze whether the selective inhibition of MMP-2/9 improves postischemic liver damage and postoperative survival.


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MATERIALS AND METHODS
 
Hepatic I/R
In female C57BL6 mice (Charles River, Sulzfeld, Germany), the left carotid artery was catheterized for measurement of arterial pressure and application of fluorescence dyes under inhalation anesthesia with isoflurane-N2O (FiO2 0.35). A warm ischemia of the left liver lobe was induced for 90 min by reversible clamping of the supplying vessels [10 , 11 ]. Intravital microscopic analyses were performed after 30 and 120 min of reperfusion. Tissue and blood samples were taken at the end of experiment after 140 min of reperfusion. All experiments were performed according to the German legislation for protection of animals.

Intravital fluorescence microscopy
The hepatic microcirculation was analyzed by an epi-illumination technique using an intravital fluorescence microscope (Leitz, Wetzlar, Germany) as described previously [10 11 12 ]. Leukocytes were labeled by an intravenous (i.v.) application of rhodamine 6G (0.1 ml, 0.05%, Sigma-Aldrich, Deisenhofen, Germany) and visualized in postsinusoidal venules and sinusoids using an N2 filter block (Leitz). All videotaped images were evaluated using CAPIMAGE® software (Zeintl, Heidelberg, Germany). In an attempt to evaluate the severity of I/R-induced perfusion injury, sinusoidal perfusion was analyzed within 5–7 acini after i.v. application of fluorescein isothiocyanate-labeled dextran (0.1 ml, 5%, Sigma-Aldrich) using an I2/3 filter block (Leitz). In each visualized acinus, the total number of sinusoids as well as the number of nonperfused sinusoids within the same acinus were counted.

T cell recruitment was analyzed in hepatic sinusoids in a separate set of experiments. CD4+ T cells were isolated from spleens of syngeneic mice by magnetic cell sorting (MiniMACS®, Miltenyi Biotec, Bergisch-Gladbach, Germany) and labeled with carboxyfluorescein succinimidyl ester (CFSE) in vitro as described [13 ]. After 30 min of reperfusion, 107 CD4+ T cells were infused and visualized in the microcirculation using an I2/3 filter block.

Immunostaining for CD45, Ly6G, and CD62P
Paraffin sections (6 µm) were quenched with 0.5% H2O2 methanol solution to block production of endogenous peroxidase, digested with pronase E, incubated in 1.5% goat serum to block nonspecific binding, and later incubated with primary antibodies. For leukocyte staining, sections were incubated with a rat anti-mouse CD45 or Ly-6G monoclonal antibody (Becton Dickinson, Heidelberg, Germany) as a primary antibody and stained with an immunohistochemistry kit (Super Sensitive link-label immunohistochemical detection system, Biogenex, San Ramon, CA). Leukocytes extravasated into the parenchymal tissue were counted in 10 high-power fields (HPF=0.09766 mm2 at microscope magnification x400) and expressed as number of cells per square millimeter of liver surface [14 ]. All cell counts were performed in a blinded manner.

For CD62P staining, sections were incubated with a rabbit anti-mouse CD62P antibody (Becton Dickinson) and stained with peroxidase immunohistochemistry kits (Vectastain, Camon, Wiesbaden, Germany). An easily detectable reddish-brown-colored end product was obtained by development in H2O2/3-amino-9-ethylcarbazol. The sections were counterstained with Mayer’s hemalaun. In each experimental group, six sections from six individual animals (10 observation fields per section) were examined by light microscopy (magnification x400), and CD62P expression was analyzed semiquantitatively in a blinded manner by using a grading system of 0–2: 0, no staining; 1, weak staining; 2, strong staining [12 ].

Deoxyuridine triphosphate-digoxenin nick-end labeling (TUNEL)
Paraffin sections were stained by terminal deoxynucleotidyl transferase-mediated TUNEL using a commercially available kit (Roche-Boehringer Mannheim Co., Germany). TUNEL-positive cells were counted in a blinded manner using light microscopy (magnification 400x) in 10 HPF.

Gelatine zymography for MMP-2 and -9 activity
Frozen samples of liver tissue (1–2 mm3) were homogenized with 300 µl homogenization buffer [20 mM Tris/HCl 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, and 0.5% sodium dodecyl sulfate (SDS)]. Protease inhibitor phenylmethylsulfonyl fluoride (100 µg/ml, Sigma-Aldrich) was added to prevent protein degradation. After homogenization, the samples were incubated at 4°C for 30 min, sonicated for 10 s, and spun at 13,000 rpm for 20 min at 4°C to remove insoluble material from the homogenate. For the zymographic analysis of gelatinases MMP-2 and MMP-9, polyacrylamide gel electrophoresis was performed on 7.5% polyacrylamide 0.5% gelatine gels (Sigma-Adrich). The following nonreducing zymography was made according the procedure described [15 ]. To avoid a dissociation of the inhibitor-enzyme complex, the tissue was powdered with a mortar at –78°C, and homogenization was performed with homogenization buffer at 4°C. There was no boiling of the samples before application of the samples to the SDS gels. The electrophoresis was run at 40 mA with a cooling to 4°C. To visualize the enzymatic digestion, the gels were stained with Brilliant Blue R. The lysis zones representing the enzymatic digest appeared as clear zones in the gel. Human recombinant (r)MMP-2 and MMP-9 standards (Sigma-Aldrich) were used to calibrate molecular weights. The results are presented as normed optical density (OD) relative to the MMP standards. Protein concentrations were determined routinely by a protein assay (Pierce, Rockford, IL) using bovine serum albumin as a standard.

Reverse transcriptase-polymerase chain reaction (RT-PCR) for CD62P and hepatocyte growth factor (HGF)
Total RNA was extracted from frozen liver tissue using RNeasy spin columns (Hilden, Germany). PCR amplification was performed as described previously [16 ]. The sense and antisense primers were as follows: murine (m)CD62P, 5'-GCCTTTGCC TACGACTCCAG-3' and 5'-GTCAAGGTACCGAAGGGATC-3'; mHGF, 5'-CAGTGCTGTGAATGAGACTGATG-3' and 5'-CACTTGACACGTCACACTTGG-3'; murine glyceraldehyde 3-phosphate dehydrogenase (mGAPDH), 5'-TGCCATTTGCAGTGGCAAAGTGG-3' and 5'-TTGTCATGGATGACCTTGGCCAGG-3'. Samples of the amplified products were analyzed on a 1% agarose gel, stained with ethidium bromide, visualized by ultraviolet illumination, and analyzed using Bio-1D software (LTF-Labortechnik, Wasserburg, Germany).

Liver enzymes
Blood samples were taken from the carotid artery at the end of the experiment (60 min of reperfusion), immediately centrifuged at 2000 g for 10 min, and stored at –80°C. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured with an automated analyzer (Hitachi 917, Roche-Boehringer Mannheim Co.) using standardized test systems (HiCo GOT and HiCo GPT, Roche-Boehringer Mannheim Co.).

Enzyme-linked immunosorbent assay (ELISA) for TNF-{alpha}
Aliquots of frozen plasma were thawed on ice. Mouse plasma TNF-{alpha} levels were measured by a sandwich ELISA using a commercially available kit, BD OptEIATM (Beckton Dickinson). A standard curve was generated by serial dilutions of rmTNF. The results are presented in pg/ml.

Survival of animals
Animal survival was determined as described [17 ]. Briefly, vascular microclamps were placed across the pedicles of the median and left liver lobes (70% of the total hepatic mass) at the level of the hilum to achieve ischemia. After an ischemic interval of 75 min, the clamps were removed, and the caudate, right, and quadrate lobes as well as the papillary process (30% of the total hepatic mass) were resected, leaving only the postischemic tissue in place. The animals were monitored for 30 days.

Interstitial migration of leukocytes in cremaster muscle
To characterize the impact of MMP-2/9 blockade on behavior of transmigrated leukocytes, we used the technique of near-infrared-reflected light oblique transillumination (RLOT) in the cremaster muscle [18 ], which allows in vivo visualization of interstitially migrating leukocytes. The right cremaster muscle of anesthetized mice was exteriorized through a ventral incision of the scrotum. Ischemia of the cremaster muscle was induced by clamping supplying vessels for 30 min as described [19 ]. Migration velocity and migration distance of interstitially localized leukocytes were analyzed using SimplePCI software (Compix Inc. Imaging System, Cranberry Township, PA) during reperfusion.

Experimental protocols and study groups
Leukocyte recruitment and tissue injury were analyzed in a sham-operated group (n=6), a group after I/R and i.v. infusion of 200 µl phosphate-buffered saline (PBS) as a vehicle (n=6), and a group after I/R, in which the MMP-2/9 inhibitor III (0.5 mg in 200 µl PBS, Calbiochem, Darmstadt, Germany; Cat. No. 444251, U.S. Patent 6,624,144 [20 ]) was given i.v. 5 min prior to reperfusion (n=6). This MMP-2/9 inhibitor III is a synthetic cyclic decapeptide H-Cys-Thr-Thr-His-Trp-Gly-Phe-Thr-Leu-Cys-OH (also called CTTHWGFTLC or CTT peptide), which selectively inhibits MMP-2 [inhibitory concentration 50% (IC50) 10 µM] and MMP-9 activity (IC50 10 µM) but does not affect activity of other MMPs, such as MT1-MMP, MMP-8, or MMP-13 [20 ]. Moreover, the peptide has not been found to exhibit any apparent toxicity. This MMP-2/9 inhibitor has been shown in vitro to inhibit migration of tumor cells as well as human endothelial cells. In vivo, this peptide binds to tumors, suppresses tumor formation, targets tumor vasculature, and prolongs survival of tumor-bearing mice [20 , 21 ]. In addition, this inhibitor has been used to localize gelatinase activity in tissue samples using in situ zymography [22 , 23 ] and to evaluate the contribution of gelatinases in various biological processes including vasoconstriction and epithelial-mesenchymal transition [24 25 26 27 ]. Although the mechanism for how the peptide inhibits gelatinase activity is not fully understood, it is suggested that the Trp residue in the HWGF motif may bind to the hydrophobic pocket of the substrate cleft in the gelatinases and that the His residue may act as a ligand for the catalytic Zn2+ ion [20 ]. The dose used for our study has been shown previously to be effective in mice [20 ].

Recruitment of CD4+ T cells was quantified after 90 min of ischemia followed by 30 and 120 min of reperfusion in nontreated or the MMP-2/9 inhibitor-treated mice (n=5 each group).

Motility of interstitially migrated leukocytes was investigated in cremaster muscle after I/R (30 min/5–40 min) in vehicle- or MMP-2/9 inhibitor-treated animals (20 leukocytes from three experiments per group).

Survival of animals was assessed after 75 min total hepatic ischemia in vehicle- and MMP-2/9 inhibitor-treated animals. Sham-operated mice served as controls (n=9 each group).

The effect of a broad-spectrum MMP inhibition on leukocyte recruitment and postischemic tissue injury was analyzed in two additional groups undergoing hepatic I/R (90/120 min) treated with the MMP inhibitor GM6001 (Calbiochem; 100 mg/kg in 200 µl 4% carboxymethylcellulose) or with the same volume of the carrier solution as vehicle given intraperitoneally 30 min before the onset of ischemia. GM6001 is a broad-spectrum hydroxamic acid inhibitor of MMPs {MMP-1: inhibitor constant (Ki)=0.4 nM; MMP-2: Ki=0.5 nM; MMP-3: Ki=27 nM; MMP-8: Ki=0.1 nM; MMP-9: Ki=0.2 nM [27 28 29 30 ]}.

Statistics
The Kruskal-Wallis test followed by the Student-Newman-Keuls test was used for the estimation of stochastic probability in intergroup comparisons. t-test was used for two-group comparison for the analysis of leukocyte motility. Survival was evaluated and calculated using the Kaplan-Meier method with Gehan-Breslow test (SigmaStat, Jandel Scientific, Erkrath, Germany). Mean values ± SEM are given. Differences were considered significant if P < 0.05.


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RESULTS
 
MMP-2 and -9 activity in the postischemic liver
In an attempt to analyze whether MMP-2 and -9 are activated in our model of hepatic I/R and to which extent the MMP-2/9 inhibitor affects the postischemic activity of both enzymes, zymography was performed in hepatic tissue. As shown in Figure 1 , the activity of MMP-9 was enhanced significantly by ~3.5-fold in the postischemic liver as compared with sham-operated mice. This increase was abolished completely in the MMP-2/9 inhibitor-treated group and was even lower than in the sham group. In contrast, the active form of MMP-2 was not detectable in sham-operated mice as well as in mice undergoing I/R (data not shown).


Figure 1
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  		Figure 1. A representative zymogram showing the active form of MMP-9 in sham-operated mice, mice after I/R, and MMP-2/9 inhibitor-treated mice after I/R (90 min/140 min). The gelatine lytic zones were digitalized. The bar graph represents the results of the quantitative analysis. n = 6 animals per group; mean ± SEM.*, P < 0.05, versus sham-operated group; #, P < 0.05, versus I/R-vehicle group.

Leukocyte-endothelial cell interactions
In the sham-operated group, only few leukocytes were found to be rolling on and firmly attached to the endothelium in postsinusoidal venules as well as stagnant in sinusoids. In contrast, 90 min of ischemia followed by 30 and 120 min of reperfusion caused a significant increase in the number of rolling and adherent leukocytes in postsinusoidal venules, as well as in the number of leukocytes stagnant in sinusoids as compared with sham-operated animals. In the treated group, however, leukocyte rolling, permanent adhesion, and sinusoidal accumulation were attenuated significantly at both reperfusion time-points (Fig. 2 ).


Figure 2
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  		Figure 2. Numbers of leukocytes rolling (A) and adherent (B) in postsinusoidal venules as well as numbers of leukocytes intravascularly accumulated in sinusoids (C) were quantitatively analyzed using intravital video fluorescence microscopy in sham-operated mice, mice after I/R, and MMP-2/9 inhibitor-treated mice after I/R. Ischemia time, 90 min; reperfusion time (R), 30 min (solid bars) and 120 min (open bars). Rolling leukocytes were defined as cells crossing an imaginary perpendicular through the vessel at a velocity markedly lower than the center-line velocity in the microvessel. Their numbers are given as cells per second per millimeter of vessel diameter (1/s/mm). Leukocytes firmly attached to the endothelium for more than 20 s were counted as permanently adherent cells and expressed as number of cells per square millimeter endothelial surface. Leukocyte accumulation in sinusoids is presented as the number of cells per acinus. n = 6 animals per group; mean ± SEM. *, P < 0.05, versus sham-operated group; #, P < 0.05, versus I/R-vehicle group.

Leukocyte transmigration
Transmigrated leukocytes were quantified in tissue sections stained for the pan-leukocyte marker CD45 or the neutrophil marker Ly6G. Leukocyte transmigration was low under control conditions (13±3/mm2 CD45-positive cells; 13±2/mm2 Ly6G-positive cells), whereas it was increased after I/R (90/140 min) by ~12- and ninefold, respectively. Approximately 70% of transmigrated leukocytes were neutrophils. The blockade of MMP-9 resulted in a significant reduction in the number of total leukocytes as well as neutrophils as compared with the vehicle-treated group (Fig. 3 ).


Figure 3
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  		Figure 3. Transendothelial migration of total leukocytes and neutrophils was analyzed in tissue sections after 140 min of reperfusion. Microphotographs demonstrate immunostaining for the common leukocyte antigen CD45 (left panels) and staining for the neutrophil marker Ly6G (right panels) in liver tissue of a sham-operated mouse (A), a mouse after I/R (B), and a MMP-2/9 inhibitor-treated mouse after I/R (C). Extravascularly localized cells (arrows) were counted in 10 HPF at original microscope magnification x400 and expressed as number of cells per square millimeter of liver surface. n = 6 animals per group; mean ± SEM. *, P < 0.05, versus sham-operated group; #, P < 0.05, versus I/R-vehicle group.

T cell recruitment
CD4+ T cells are suggested to play an important role during hepatic I/R [31 ]. MMP-2 and -9 expression has been shown to be up-regulated in T cells upon activation [32 , 33 ]. As demonstrated in Figure 4 , the number of CD4+ T cells accumulated in sinusoids was elevated significantly in postischemic livers as compared with the sham-operated group. After 30 min of reperfusion, 12 ± 3% of intrasinusoidally accumulated CD4+ T cells were found in the perivascular space, and this percentage was elevated to 26 ± 3% after prolongation of reperfusion time, up to 120 min. Similar to neutrophils, the I/R-induced T cell accumulation was significantly lower in MMP-2/9 inhibitor-treated mice. Moreover, the percentage of transmigrated T cells was reduced to 8 ± 2% and 16 ± 6% after 30 and 120 min of reperfusion, respectively.


Figure 4
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  		Figure 4. Recruitment of CFSE-labeled CD4+ T cells in hepatic microvessels was analyzed using intravital microscopy in sham-operated mice (A), mice after I/R (B), and MMP-2/9 inhibitor-treated mice after I/R (C). Ischemia time, 90 min; reperfusion time, 30 min (solid bars) and 120 min (open bars). The arrows depict accumulated CD4+ T cells; the arrowheads mark extravascularly localized (transmigrated) CD4+ T cells. Original microscope magnification, x500. n = 5 animals per group; mean ± SEM. *, P < 0.05, versus sham-operated group; #, P < 0.05, versus nontreated I/R group.

Leukocyte motility
Leukocyte motility was analyzed in the postischemic cremaster muscle by RLOT intravital microscopy and a bioimaging software for cell tracing. Transmigrated leukocytes were visualized (Fig. 5 ), and parameters of their migration, i.e., migration velocity, curve-line, and straight-line migration distances, were measured semiautomatically by "SimplePCI" software. As shown in Figure 5 , the migration velocity and the curve-line distance were reduced by approximately twofold after MMP-9 blockade, whereas the straight-line migration distance was by approximately tenfold shorter in the treated group than in the nontreated I/R group.


Figure 5
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  		Figure 5. Interstitially migrating leukocytes (pointed by arrows) were visualized using near-infrared RLOT intravital microscopy in the postischemic (ischemia: 30 min/reperfusion: 5–40 min) cremaster muscle of vehicle-treated mice or MMP-2/9 inhibitor-treated mice. Parameters of leukocyte motility (migration velocity and curve-line and straight-line migration distance) were quantified for 20 interstitially migrating leukocytes during 5 min in digitalized intravital microscopic video sequences using the bioimaging software SimplePCI (Compix Inc. Imaging System). Migration tracks of individual leukocytes are presented in the intravital microscopic image as green lines. Original microscope magnification, x857. #, P < 0.05, versus I/R-vehicle group.

CD62P expression
Next, we investigated whether the MMP-9 inhibition influences the expression (or endothelial translocation) of the adhesion molecule CD62P. As shown by immunostaining, CD62P was expressed clearly on the endothelium of postsinusoidal venules in sham-operated mice as well as in mice undergoing hepatic I/R (Fig. 6 , left panels). In contrast, the endothelial expression of CD62P was significantly lower in postischemic livers from mice treated with the MMP-2/9 inhibitor. It was not possible to distinguish between CD62P stored in endothelial Weibel-Palade bodies and CD62P translocated to the endothelial surface. To support the immunohistochemical results, we assessed mRNA expression of CD62P by RT-PCR and observed that the postischemic up-regulation of CD62P was slightly reduced in response to the MMP-9 blockade (Fig. 6 , right panel).


Figure 6
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  		Figure 6. CD62P expression in the liver in sham-operated mice, mice after I/R, and MMP-2/9 inhibitor-treated mice after I/R (90 min/140 min). The left panels show representative immunohistochemical images of CD62P expression in postsinusoidal venules (arrows) as well as semiquantitative data using a grading system of 0–2: 0, no staining; 1, weak staining; 2, strong staining. Arrowheads indicate CD62P-positive platelets in postischemic sinusoids (not quantified). Original magnification, x400; n = 6 each group. #, P < 0.05, versus nontreated I/R group. The right panels demonstrate representative gels of RT-PCR analysis of CD62P mRNA expression in homogenates of liver tissue. The bar graphs represent the results of the semiquantitative analysis, which is given as a ratio to GAPDH. n = 3 animals per group; mean ± SEM.

TNF-{alpha} levels
CD62P translocation to the endothelial surface from Weibel-Palade bodies occurs several minutes after endothelial activation and is triggered by inflammatory mediators such as TNF-{alpha} or thrombin. In the present study, we measured systemic levels of TNF-{alpha} in plasma and observed that I/R enhanced the TNF-{alpha} levels significantly in vehicle-treated mice (363±26 pg/ml) as compared with the sham-operated group (282±12 pg/ml). This effect, however, was attenuated significantly in the I/R group undergoing MMP-9 blockade (307±12 pg/ml).

Hepatic I/R injury
Sinusoidal perfusion failure was determined as a measure of microvascular I/R injury. After I/R, the number of nonperfused sinusoids was increased significantly as compared with sham-operated mice. In mice treated with the MMP2/9 inhibitor, however, sinusoidal perfusion was not improved and did not differ from the vehicle-treated I/R group (Fig. 7A ). For assessment of hepatocellular necrotic damage of the postischemic liver, the AST/ALT serum activities were measured. After I/R, a dramatic increase in the liver enzyme activities was observed in vehicle-treated mice as compared with sham-operated mice. In contrast, the postischemic level of AST serum activity was reduced significantly in mice after MMP-9 inhibition. The postischemic level of ALT did not differ significantly from the untreated I/R group (Fig. 7B) . To analyze whether MMP-9 is involved in the manifestation of apoptotic I/R injury, TUNEL staining was performed in tissue sections. As shown in Figure 7C , the number of TUNEL-positive hepatocytes was markedly higher in the postischemic group than in sham-operated controls. Although a lower number of apoptotic hepatocytes were detected in MMP-2/9 inhibitor-treated mice, these changes did not reach the level of significance.


Figure 7
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  		Figure 7. Microvascular and hepatocellular injury was analyzed in sham-operated mice, mice after I/R, and MMP-2/9 inhibitor-treated mice after I/R. (A) Sinusoidal perfusion was measured as a parameter of microvascular hepatic I/R injury. Microvascular perfusion failure is presented as percentage of nonperfused sinusoids (nonperfused sinusoids/total sinusoids of an acinusx100%). Ischemia time, 90 min; reperfusion time, 30 min (solid bars) and 120 min (open bars). (B) Serum activity of the liver enzymes AST (solid bars) and ALT (shaded bars) was determined as a marker of hepatocellular necrotic injury after 90 min of ischemia followed by 140 min of reperfusion. (C) TUNEL-positive hepatocytes were quantified as a parameter of apoptosis in 10 HPF at original microscope magnification x400. n = 6 animals per group; mean ± SEM. *, P < 0.05, versus sham-operated group; #, P < 0.05, versus I/R-vehicle group.

HGF mRNA expression
Expression of the regenerative factor HGF was determined in an attempt to evaluate the impact of MMP-9 blockade on early regeneration of the postischemic liver. However, no differences in hepatic HGF expression were detected between the sham-operated group and both postischemic groups after 140 min of reperfusion (Fig. 8 ).


Figure 8
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  		Figure 8. HGF mRNA expression was analyzed as a parameter of early regeneration using RT-PCR in homogenates of liver tissue from sham-operated mice, mice after I/R, and MMP-2/9 inhibitor-treated mice after I/R (90 min/140 min). The bar graph represents the results of the semiquantitative analysis, which is given as a ratio to GAPDH. n = 3 animals per group; mean ± SEM.

Animal survival
Animal survival is a well-recognized parameter for evaluation of the long-term effectiveness of treatment strategies applied in experimental studies. In the sham-operated group, all animals survived 30 days after liver resection, whereas seven of nine animals subjected to 75 min of total ischemia died within 24 h of surgery. In contrast, survival was improved significantly in the group after MMP-9 inhibition, as only two mice died during the first 24 h and one mouse after 4 days after ischemia (Fig. 9 ).


Figure 9
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  		Figure 9. Survival of animals undergoing 75 min of total hepatic ischemia followed by resection of nonischemic tissue without treatment ({circ}) and after treatment with MMP-2/9 inhibitor (dashed line and •) as compared with the sham-operated group (solid line and •). No significant difference in the number of animals surviving hepatic ischemia was observed between the sham-operated and the treated I/R group. n = 9 animals per group; mean ± SEM. *, P < 0.05, versus sham-operated group; #, P < 0.05, versus I/R-vehicle group.

Effect of a broad-spectrum MMP inhibitor on leukocyte recruitment and I/R injury
To corroborate the results described above, we also tested the broad-spectrum MMP inhibitor GM6001, a compound that is structurally different from the used MMP-2/9 inhibitor. Broad-spectrum MMP inhibition resulted in a significant reduction of intrasinusoidal leukocyte accumulation and leukocyte/neutrophil transmigration, whereas leukocyte rolling was not affected (Table 1 ). In contrast to the data after selective MMP-2/9 inhibition, postischemic sinusoidal perfusion was improved significantly at both reperfusion time-points. The increase in the ALT/AST activity, however, did not differ between both postischemic groups.


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  		Table 1. Effect of a Broad-Spectrum MMP Inhibitor on Postischemic Leukocyte Recruitment and I/R Injury


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DISCUSSION
 
MMP-2 and -9 are involved in the degradation of ECM components and are expressed not only in nonparenchymal liver cells (i.e., Kupffer cells and sinusoidal endothelial cells) but also in different subsets of leukocytes (T cells, neutrophils, monocytes, macrophages). As they can potentially destroy the components of the basal membrane and ECM, we hypothesized that these enzymes play a critical role in the process of leukocyte migration from microvessels to the sites of inflammation during hepatic I/R. We used a specific MMP-2/9 inhibitor in the first instance to determine the potential role of these enzymes in regulating interactions of leukocytes with endothelial cells and ECM in the postischemic liver. The analysis of MMP-2/9 activity, however, revealed that only MMP-9 is activated in response to I/R. In contrast, MMP-2 activity was not detectable under basal conditions as well as after I/R. In line with our findings, recent studies have shown that MMP-9 is up-regulated and activated quickly during hepatic I/R in rats as well as after transplantation of human liver, whereas MMP-2 is not activated during the first 48 h of reperfusion and expressed constitutively at low level [3 , 34 ]. The second important result given by zymography is that the postischemic increase in MMP-9 activity was blocked completely by the MMP-2/9 inhibitor used. It is noteworthy that the zymography method detects active MMP forms but does not allow distinguishing whether this effect is a result of direct binding of the inhibitor on MMP-9 or to other mechanisms (i.e., involvement in the MMP-9 activation process or inactivation through MMP tissue inhibitors).

Leukocyte migration from blood into tissues involves a series of molecular interactions between leukocyte and endothelial cells. This allows leukocyte rolling, firm arrest, and transendothelial migration to occur. In the present study, we have shown that MMP-9 blockade affects all steps of the leukocyte migration process. As well known, leukocyte rolling in the postischemic liver is mediated by CD62P [11 , 35 ], an adhesion molecule, which is functionally expressed by extrusion to the cell surface upon endothelial activation [36 ]. Translocation of CD62P is initiated by thrombin and inflammatory mediators, particularly by TNF-{alpha}. Gearing et al. [5 ] reported that MMP blockade prevents the processing of the TNF precursor and the rise in blood levels of TNF-{alpha}, which is strongly released already during early reperfusion. Therefore, we assumed that MMP-9 inhibition attenuates leukocyte rolling in venules by affecting TNF-{alpha} release and CD62P translocation. In fact, we observed that the I/R-induced increase in the systemic levels of TNF-{alpha} as well as the endothelial expression of CD62P in postsinusoidal venules were lower in response to MMP-9 blockade. Although these data represent an association rather than a causal relationship among MMP-9 activation, TNF-{alpha} release, and CD62P expression, they might indicate a possible link between MMP-9 inhibition and the changes in leukocyte migration.

The next step following leukocyte rolling is their permanent adherence. The effect of MMP-2/9 inhibition on leukocyte adherence in postsinusoidal venules might be a result of the attenuation of leukocyte rolling. Intrasinusoidal leukocyte accumulation is, however, promoted directly by MMP-2 and -9, as it does not require the rolling phase. A recent in vitro study has shown that MMP-2 and -9 convert big endothelin-1 [1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 ] to endothelin-1 [1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 ], which up-regulates the expression of adhesion molecules (CD54, CD62E, CD62L) on coronary artery endothelial cells and ß2-integrins (CD11b/CD18) on the surface of neutrophils, leading to an increased attachment of neutrophils to the endothelium [37 ]. Our intravital microscopic data support these in vitro results, as leukocyte accumulation in hepatic sinusoids was clearly attenuated in the treated group.

Leukocyte transendothelial migration is a critical step in the process of leukocyte recruitment. Not only the mechanisms of transendothelial migration but also the pathophysiological role of transmigrated leukocytes remain not fully understood. In a recent study, we have shown that neutrophil transendothelial migration in the postischemic liver is mediated by junctional adhesion molecule-A (JAM-A), expressed in endothelial tight junctions [14 ]. However, beyond the endothelial barrier, transmigrated leukocytes have to cross the basement membrane before migrating into the perivascular space, and this requires proteolytic disruption of ECM components. In the present study, we demonstrate that the pharmacological inhibition of MMP-9 attenuates transmigration of neutrophils during hepatic reperfusion. This effect can be explained by the ability of gelatinases to destroy collagen IV of the basement membrane, which enables extravasation, as well as by the impact of MMP-9 inhibition on the steps preceding transmigration, rolling, and firm adherence. Moreover, gelatinases control and directly modulate activity of chemokines, a group of chemotactic molecules, which specifically attract and recruit immune effector cells (including neutrophils and T cells) to sites of injury [38 ].

The effect of MMP-9 blockade on motility of interstitially migrating leukocytes in postischemic tissue was analyzed in the cremaster muscle by RLOT intravital microscopy. This assay allows high-quality and pseudo three-dimensional visualization of migrating leukocytes in the perivascular space without use of fluorescence dyes. Indeed, transmigrated leukocytes in the treated group were characterized by markedly reduced migration velocity and migration distances. This points to an important role of MMP-9 for the destruction of ECM components during leukocyte interstitial migration and regulation of leukocyte chemotaxis during postischemic hepatic inflammation. The stronger impact of MMP-9 blockade on straight-line migration distance than on curve-line distance suggests that leukocytes lose the ability to "target" migration in response to MMP-9 blockade and move in tissue rather chaotically.

CD4+ T cells have been shown to contribute to the antigen-independent, hepatic I/R damage [31 ]. Transmigrating T lymphocytes secrete proteolytic enzymes, in particular, MMP-2 and MMP-9, to pass through the subendothelial basement membrane en route to the perivascular tissue. Recent studies have reported that MMPs regulate transendothelial passage of lymphocytes across high endothelial venules from the blood into lymph nodes [39 ] and that cytokine-induced migration of T cells in a membrane filter migration chamber is reduced after a selective MMP-2/9 inhibition [40 ]. In our study, we have shown for the first time in vivo that MMP-9 is required for accumulation and transmigration of CD4+ T cells during I/R-induced, antigen-independent hepatic inflammation.

In patients after liver transplantation, the postoperative level of AST correlated with MMP-9 level in serum [34 ]. Inhibition of MMP activity with a broad-spectrum inhibitor significantly decreased liver injury in postischemic liver tissue as assessed by histological studies and serum hepatic enzyme level [3 ]. Moreover, disassembly of cold-preserved hepatic endothelilal cells from MMP-9–/– mice is attenuated as compared with wild-type cells [41 ]. In the present study, we analyzed the impact of MMP-9 inhibition on microvascular and hepatocellular necrotic/apoptotic I/R injury during initial reperfusion (140 min), early regeneration, and animal survival. We demonstrated that selective blockade of MMP-9 exerts a weak, protective impact on early I/R injury. Although sinusoidal perfusion failure and apoptotic injury were not improved significantly, only AST, but not ALT activity, was attenuated in response to MMP-9 blockade. Furthermore, mRNA expression of HGF, a factor of early regeneration produced by nonparenchymal liver cells, did not differ between treated and untreated groups. Despite a weak protection from I/R injury during early reperfusion, MMP-9 inhibition significantly improved postoperative animal survival. In line with our findings, Solorzano et al. [30 ] reported that a broad-spectrum MMP inhibition with GM-6001 is not able to reduce and even exacerbates endotoxin as well as concavalin A-induced liver injury but clearly prevents mortality. Apparently, the interruption of MMP-9-mediated leukocyte-matrix interactions attenuates tissue injury more effectively during the later reperfusion phase. The present findings complement our study in JAM-A–/– mice, in which increased intravascular adherence of neutrophils as well as accumulation of T cells were associated with aggravated hepatic I/R injury despite attenuation of neutrophil transmigration [14 ].

Finally, we compared the effects of the MMP-2/9 inhibitor with those of a widely used, broad-spectrum MMP inhibitor (GM-6001) on leukocyte migration and tissue injury. It is interesting that we noticed some differences in the extent of neutrophil emigration and liver enzyme activity between the I/R-vehicle groups in both sets of experiments. These discrepancies might be explained by different chemical composition of the vehicle solutions as well as by different routes and timing of vehicle application. Comparing the effects of both inhibitors on leukocyte migration, we observed that the broad-spectrum MMP blockade does not affect leukocyte rolling, has a weaker effect on leukocyte adherence, but reduces neutrophil transmigration in a similar manner as observed after MMP-9 blockade. The fact that the effect of the general MMP inhibition was not stronger than that after selective MMP-9 inhibition is interesting and warrants further exploration. Based on the current discussion in the literature about a dual role of MMPs during inflammation [38 ], we can only speculate that some members of the MMP family may mediate, not only functions of proinflammatory cytokines but also of those that are involved in the reduction of inflammatory reactions (e.g., IL-10, IL-12, IL-4). Indeed, MMP-2 and -3, which are blocked by GM6001, exert protective effects during arthritis [42 , 43 ].

Taken together, we have shown that MMP-9 but not MMP-2 is activated in the postischemic liver. MMP-9 is involved in the early recruitment steps of neutrophils and CD4+ T cells, promotes the process of their transendothelial migration during hepatic I/R, and is required for motility of interstitially migrating leukocytes. A pharmacological MMP-9 inhibition is associated with attenuation of TNF-{alpha} release and endothelial CD62P expression, weakly protects from early microvascular, necrotic, and apoptotic hepatocellular I/R damage, but improves postoperative survival.


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ACKNOWLEDGEMENTS
 
The authors thank Mrs. C. Csapo and Mrs. A. Schropp for technical assistance (Institute for Surgical Research, University of Munich) and Dr. Rauch (Institute of Clinical Chemistry, University of Munich) for measuring ALT/AST activities. The study was supported by research grants from Deutsche Forschungsgemeinschaft (FOR440/2), EU FP6 NoE "MAIN" (LSHG-CT-2003-502935), and Friedrich-Baur Stiftung.

Received August 23, 2005; revised January 17, 2006; accepted February 14, 2006.


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