The FASEB Journal express article 10.1096/fj.01-0279fje. Published online November 14, 2001.
Rat liver injury following normothermic ischemia is prevented by a phosphinic matrix metalloproteinase inhibitor Raffaele Cursio,* Bernard Mari,† Krystel Louis,† Philippe Rostagno,‡ Marie-Christine SaintPaul,§ Jean Giudicelli,¶ Virginie Bottero,† Patrick Anglard,** Athanasios Yiotakis,†† Vincent Dive,‡‡ Jean Gugenheim,* and Patrick Auberger† *Laboratoire de Recherches Chirurgicales, IFR50, Faculté de Médecine Pasteur, Nice, France; † INSERM U526, IFR50, Faculté de Médecine Pasteur, Nice, France; ‡Centre Antoine Lacassagne, Nice, France, France; §Service d'Anatomo-Pathologie, Hôpital Pasteur, Nice, France; ¶Laboratoire de Biochimie, Faculté de Médecine, Nice, France; **Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM U184, Illkirch, France; ††Department of Chemistry, Laboratory of Organic Chemistry, University of Athens, Athens, Greece; ‡‡CEA, Département d’Ingénierie et d’Etudes des Protéines, CEA-Saclay, France. Corresponding author: Bernard Mari, INSERM U526, IFR50, Faculté de Médecine Pasteur, Nice, France. E-mail:
[email protected] R. Cursio and B. Mari contributed equally to this work. ABSTRACT Hepatic ischemia occurs in liver transplantation, hemodynamic or cardiogenic shock, and liver resection associated with trauma or tumor. Ischemia/reperfusion (I/R) injury results in microcirculation failure followed by apoptosis and necrosis. Matrix metalloproteinases (MMPs) are involved in many physiological and pathological processes, but their expression and function during liver I/R remains poorly documented. In this study, we evaluated the expression of nine MMPs and their natural inhibitors, tissue inhibitors of MMPs (TIMPs), in a rat model of liver I/R. Analysis of MMP and TIMP expression show that although most of these genes are not constitutively expressed in the normal liver, they are induced in a specific time-dependent manner following I/R. Stromelysin-1, gelatinase B, and collagenase-3 are induced during the early phase of acute liver injury associated with inflammation and increased necrosis/apoptosis, whereas gelatinase A, membrane type-MMP, stromelysin-3, metalloelastase, TIMP-1, and TIMP–2 are essentially detectable during the recovery phase of liver injury corresponding to hepatocyte regeneration. This observation suggested that MMPs and TIMPs could play both deleterious and beneficial roles following I/R. We thus tested the effect of a specific phosphinic MMP inhibitor on acute liver I/R injury. Inhibition of MMP activity was shown to significantly decrease liver injury in ischemic/reperfused liver tissue as assessed by histological studies and serum hepatic enzyme levels. We therefore propose that MMP inhibitors may be of clinical relevance in liver-associated ischemic diseases or after liver transplantation.
Key words: matrix metalloproteinases (MMPs) • tissue inhibitors of matrix metalloproteinase (TIMPs) • Ischemia/reperfusion • necrosis • apoptosis
D
egradation of extracellular matrix (ECM) is essential in many developmental and physiological processes, including angiogenesis, branching morphogenesis, and wound healing. This degradation involves expression and activation of a set of specific matrix metalloproteinases (MMPs). MMPs define a family of more than 20 members collectively capable of degrading all components of the ECM (1–3). They are Zn2+- and Ca2+-dependent endopeptidases that are synthesized as secreted or transmembrane proenzymes and processed to an active form by removal of an amino-terminal prodomain. According to their substrate specificity and structural similarities, MMPs can be classified into several subgroups, such as collagenases, degrading fibrillar collagen, gelatinases that have high activity against denatured collagen, stromelysins that degrade noncollagen components of the ECM, membrane-type MMPs, and other MMPs that are less characterized. Although these enzymes have been involved in tissue remodeling processes, their participation in cell migration, cell death, and cell survival as well as their modulation of biologically active molecules by cleavage or release from the ECM have also been recently demonstrated (3). The activity of all MMPs is regulated at several levels, including gene transcription, proenzyme activation, and inhibition of active enzymes by a family of endogenous inhibitors, the tissue inhibitors of MMPs (TIMPs), which comprise at least four members (TIMP 1–4) (2–4). Recent studies have indicated that matrix degradation is essential for liver repair events. In particular, it has been shown that an imbalance between matrix synthesis and degradation is involved in hepatic fibrogenesis (for review, see ref 5). Indeed, hepatic fibrogenesis is accompanied by an elevation of TIMP-1 and TIMP-2 levels and a decrease in global collagenolytic activity (6–9). However, this global down-regulation of MMP expression is associated with an increased expression of two MMP members, gelatinase A and MT1-MMP (10, 11), indicating that liver fibrosis results in profound changes in the regulation of matrix degradation. Among the different cell types in the liver, hepatic stellate cells seem to play an essential role in this pathological process (12). Matrix remodeling also takes place during liver regeneration. It has been shown that stromelysin-1 and TIMP-1 were induced in a distinct time frame during liver regeneration in rats treated with carbon tetrachloride (6, 7, 13). Regeneration after partial hepactectomy is also followed by a dramatic increase in gelatinase A and B and by an increase in MT1-MMP and TIMP-1 (14, 15). Moreover, it has been shown that a single liver injury induced by carbon tetrachloride, which is characterized by hepatocellular necrosis and inflammation before liver regeneration, results in profound changes in MMP and TIMP levels during the acute phase of injury (15). Hepatic ischemia occurs in a variety of circumstances, including liver transplantation, hemodynamic or cardiogenic shock, and liver resection for trauma or tumor. This injury results in microcirculation failure followed by necrosis, apoptosis, and finally liver regeneration. All these processes involve matrix breakdown and resynthesis and migration of inflammatory cells, which both require expression of MMPs. Hepatocyte injury following reperfusion is partly dependent on Kupffer cell activation and production of TNF-α, and it occurs in two distinct phases. The first, early, phase of injury occurs 1–3 h after reperfusion, depends on activated
Kupffer cells, and is mainly mediated by oxygen radicals. The second, later, phase of injury occurs at 6–24 h after reperfusion, and it is associated with neutrophil sequestration and is dependent on both TNF-α and the presence of neutrophils. Thus, the sequence of events during reperfusion injury may be that reperfusion triggers the release of oxygen radicals, causing Kupffer cell activation, which in turn results in increased TNFα- production. TNF-α initiates neutrophil accumulation and release of additional reactive products (16). Kuppfer cells have the capacity to secrete MMPs (17, 18). Several studies have reported a role for MMPs in cold preservation injury of the liver in humans and in rats (19), and several compounds from liver preservation solutions actually behave as cryptic inhibitors of MMPs (20). Nevertheless, no data concerning the expression and the potential function of MMPs and TIMPs during normothermic liver ischemia/reperfusion (I/R) are currently available. However, the expression of a few MMPs has been recently described in other organs in response to ischemia. Notably, it has been shown that gelatinases A and/or B and in some cases stromelysin-1 are rapidly induced in different models, including lung, heart, and cerebral ischemia (21–26). This early induction correlates with acute organ injury and strongly suggests that MMP inhibition could improve organ recovery. Indeed, it has been shown that gelatinase B inhibition, with a specific neutralizing antibody, reduced infarct size after cerebral focal ischemia (21) and that doxycycline, a tetracycline-derived molecule that can inhibit MMP activity, improved heart mechanical function recovery following ischemia (22). However, the expression of other MMP members and their precise functions in normothermic I/R are not known. To further investigate the potential function of MMPs and TIMPs during liver I/R, we evaluated the expression of nine MMPs and three TIMPs during the reperfusion phase after normothermic liver ischemia in rats. Gelatinases A and B, and also stromelysin-1 and matrilysin, may indeed contribute to liver remodeling by altering the liver basement membrane. Moreover, the rat interstitial collagenase (collagenase-3) and MT1-MMP are known to cleave fibrillar collagen (2), which is particularly abundant in the Disse space. Moreover, MT1-MMP and stromelysin-1 have been involved in the activation of other MMPs, notably gelatinase A and B (1). Expression patterns were studied during both the acute phase, characterized by inflammation and hepatocellular necrosis/apoptosis, and the regeneration phase, resulting in the replacement of damaged hepatocytes. We then tested the effect of a phosphinic MMP inhibitor on the early acute injury induced by liver I/R. Our results show that MMP inhibition significantly decreases liver necrosis and apoptosis in ischemic/reperfused liver tissue as assessed by histological studies and serum hepatic enzyme levels. MATERIALS AND METHODS Animal preparation and liver ischemia procedure All experiments were conducted in accordance with institutional guidelines for the care and use of laboratory animals. Male Wistar rats (weighing 200 g) were purchased from the CNRSCNSEAL (Orleans La Source, France). The rats, housed individually, were allowed free access to food and water before, during, and after liver ischemia. The animal rooms were windowless with controlled temperature (22±2°C) and lighting (14 h light/10 h dark). The animals were subjected to a hepatic ischemia procedure, as described previously (27). In brief, under ether
anesthesia, a segmental normothermic ischemia of the liver was induced by occluding the blood vessels to the median and lateral lobes. This procedure is considered to render ischemia in 70% of liver tissue. The abdomen was closed in two layers with 2-0 silk, and the animals were housed individually in plexiglass cages. After 90 min, the animals were reanesthetized and the vascular clamp was released. At different times after the surgical procedure, the animals were anesthetized and killed by exsanguination, and both ischemic and normal liver lobes were collected and frozen in liquid nitrogen. cDNA probes cDNAs used to detect MMP-2, -3, -7, -9, -10, -11, -13, and -14 and TIMPs 1–3 were previously described (28, 29). cDNA for MMP-12 was a kind gift of Dr. Sophie Tartare-Deckert. RNA extraction and Northern blot analysis Tissues were homogenized, and total RNAs were extracted using a Tripure kit (Roche, Basel, Switzerland). RNAs (15 µg) were subjected to electrophoresis on a 1% agarose gel and transfered to Hybond N+ membranes (Amersham Pharmacia Biotech, ). The different probes were labeled with α[32P] dATP (Amersham) following the Strip-EZ kit (Ambion, Austin, TX) procedure. Hybridizations were carried out at 68°C for 18 h (ULTRAhyb, Ambion). Membranes were washed twice for 10 min at 65°C in 2× SSC containing 0.1% sodium dodecylsulfate (SDS) and once for 10 min at 65°C in 0.1 SSC containing 0.1% SDS. Membranes were analyzed after a 24-h exposure by using a phosphoimager system (Storm 840, Molecular Dynamics, Sunnyvale, CA). Transcripts were quantified using Imagequant software (Molecular Dynamics). Membranes were stripped following the Strip-EZ kit protocol and reused four to six times. Substrate gel analysis (zymography) :iver tissue (100 mg) was homogenized and solubilized at 4°C in 10 ml of extraction buffer containing 50 mM Tris/HCl (pH 7.4), 20 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 100 µg/ml leupeptin. Samples were centrifuged, the protein content of the supernatants was measured with a Bio-Rad (Hercules, CA) assay, and 100-µg extracts were analyzed by gelatin zymography. In brief, proteins were electrophoresed by SDS-PAGE on a 10% polyacrylamide gel containing 0.3% gelatin in the absence of reducing reagents. After a 2-h treatment with 2.5% Triton X-100, gels were incubated in 50 mM Tris/HCl (pH 7.4) containing 150 mM NaCl and 1mM CaCl2 at 37°C for 24 h in the presence or the absence of 20 µM phosphinic MMP inhibitor RXPO3. In vivo experiments Rats were divided into four groups and were subjected to liver I/R procedure, as previously described (27). Groups 1 and 2 (four animals in each) were injected intravenously with 0.5 ml of a phosphate-buffered saline (PBS) solution containing 0.5% dimethyl sulfoxide (DMSO) via portal vein 2 min before induction of liver ischemia and 2 min after release of the clamp. Groups 3 and 4 (six animals in each) were injected at the same time with 0.5 ml of the phosphinic MMP inhibitor RXPO3 RI (30) at 0.5 mg/ml in PBS containing 0.5% DMSO. Animals in groups 1 and
3 and in 2 and 4 were sacrificed 6 and 24 h following reperfusion, respectively, and samples of serum and liver (normal and ischemic lobes for each animal) were collected and frozen at –80°C. Measurement of serum aminotransferases and lactate dehydrogenase levels Blood samples for measurement of serum aspartate (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) were collected from indwelling suprahepatic catheter and quantitated using standard clinical automated analysis. Histological studies Specimens were fixed in 10% formalin and embedded in paraffin. Sections at 3-µm intervals were stained with hematoxylin and eosin. The extent of liver necrosis was semiquantitatively assessed in nine different samples of ischemic lobes for each rat (four rats/group). The degree of damage to the liver was determined as a percentage of total area that exhibited hepatocellular necrosis. Blind analysis was carried out for all histological studies. To detect apoptotic cells, 5-µm sections were prepared from cryopreserved liver tissue (four rats/group, two samples for each animal corresponding to ischemic or control lobes) for the terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end labeling (TUNEL) method, as previously described (27). In brief, the sections were fixed with 4% paraformaldehyde in PBS for 30 min. After two washes in PBS buffer, tissue sections were incubated on ice for 2 min in permeabilization solution (0.1% Triton X-100 in PBS). After two washes in PBS, 50 µl TUNEL reaction mixture (Boehringer-Mannheim) was added to samples and incubated in a humidified chamber for 1 h at 37°C. Sections were washed twice in PBS. After the addition of propidium iodide and two final washes in PBS, sections were dried, treated with an antifading solution, and examined by a confocal microscopy (Leica Microsystems, Bannockburn, IL) at ×40 power field. For each section, eight fields were randomly chosen. Eight hundred to 1000 nuclei were examined, and TUNEL-positive hepatocytes were counted. The number of TUNEL-positive hepatocytes per 100 hepatocytes was calculated. Western blot analysis Liver tissue was homogenized in 50 mM Tris/HCl (pH 7.4) containing 25 mM NaCl, 5 mM EDTA, 0.2% Triton X-100, 1 µg/ml pepstatin, 10 µg/ml leupeptin, and 10 g/ml aprotinin. Homogenates were centrifuged at 14,000 g at 4°C for 20 min. Soluble extracts (150 µg/lane) were resolved by 10% SDS-PAGE under reducing conditions. After they were transferred to immobilon membrane (Millipore, Bedford, MA), the membranes were blocked for 2 h at room temperature with 3% (w/v) bovine serum albumin and probed overnight with a murine antiPARP antibody (mAb SA250, BIOMOL Research Laboratories, Plymouth Meeting, PA). Blots were incubated with the secondary antibody anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA), and immunoreactivity was revealed by enhanced chemoluminescence.
Statistical analysis The comparison between samples from control and treated rats for statistical significance was performed according to a paired Student’s t test for serum enzyme levels and histological analysis. Statistical significance was set at P < 0.05. Errors bars in figures represent standard errors. RESULTS Expression of MMP- and TIMP-specific transcripts following liver I/R Using a model of partial liver I/R in rats (27), liver expression of MMPs and TIMPs--namely gelatinase A and B, stromelysin-1–3, collagenase-3, matrilysin, metalloelastase, membrane-typeMMP (see Table 1 for nomenclature), and TIMP-1–3--was evaluated by Northern blot analysis (Fig. 1A). In normal and nonischemic liver lobes, expression of most MMPs and TIMPs was low or undetectable, except for gelatinase A and MT1-MMP. Following liver I/R, MMP and TIMP expression was induced in a specific time-dependent pattern (Fig. 1A). The transcripts for collagenase-3 and stromelysin-1 were rapidly induced 3 h following reperfusion and returned to basal levels after 24–48 h. Gelatinase B presented a similar profile but with a biphasic induction that was maintained 96 h after I/R. These three MMPs are the main members expressed during the early phase of liver injury. After 24–48 h, metalloelastase and stromelysin-3 were induced, and their expression remained elevated even after 1 wk, particularly in the case of stromelysin-3. MT1-MMP expression and gelatinase A expression were indistinguishable with a low constitutive level and a peak 2–3 days after I/R, followed by a slow decline. Transcripts encoding for matrilysin and stromelysin-2 were not detectable in our conditions (data not shown). TIMP-1 and -2 mRNAs presented a similar pattern of induction, increasing after 24 h, being maximal at 48 h, and then declining at 72 h. The TIMP-3 transcript was undetectable in basal or in ischemic conditions. Gelatinolytic activity in liver tissue following I/R To verify that induction of MMP mRNAs correlated with an increase in expression and activity of the corresponding proteins, gelatin zymography was performed in normal and ischemic liver lobes in the same conditions (Fig. 1B). This technique allows the detection of the two gelatinases with enzymatic activity at approximatively 82–92 kDa for gelatinase B and 62–72 kDa for gelatinase A. This gelatinolytic activity was abrogated by incubation with 20 µM of the MMP inhibitor RXPO3 (data not shown). The basal gelatinolytic activity was low for gelatinase B in normal liver and increased rapidly after ischemia, with a biphasic profile reflecting the accumulation of MMP-9 mRNA (compare Fig. 1A and B). The activity then decreased at 72 h and returned to basal level 1 wk after I/R. It is of interest that active gelatinase B at 82 kDa was detectable only in ischemic conditions, indicating that the protein was induced and activated following I/R. In the case of gelatinase A, gelatinolytic activity was poorly detected until 48 h, and both the zymogen (72 kDa) and the mature form (62 kDa) increased after this time and remained stable during the reperfusion period. In each case, transcript and protein patterns were superposable, with a strong induction of gelatinase B during the acute phase of liver injury and an increase in gelatinase A level and activity during liver regeneration.
Administration of a MMP inhibitor decreases serum aminotranferases and lactate dehydrogenase levels in liver I/R-treated rats To evaluate the effect of MMP inhibition on ischemia-induced liver injury, rats were injected with 0.5 mg of the MMP inhibitor RXPO3 (30) or vehicle alone (2% DMSO in PBS) twice two min before the induction of ischemia and 2 min after clamp release in order to inhibit both constitutive liver MMPs and those induced during the course of reperfusion. Blood samples were collected from animals 6 and 24 h following ischemia, and serum levels of AST, ALT aminotranferases, and LDH were measured (Fig. 2). As expected, AST, ALT, and LDH levels increased 6 h (AST: 2550±1269 UI/L, ALT: 912±475 UI/L, and LDH: 2844±763 UI/L) and 24 h (AST: 20038±4847 UI/L, ALT: 10975±3230 UI/L, and LDH: 117850±23514 UI/L) after reperfusion. In control and sham-operated animals, the serum levels were 85±15 UI/L for AST, 37±29 UI/L for ALT, and 975±100 UI/L for LDH. It is of interest that the release of liver enzyme was slightly diminished in animals treated with RXPO3 after 6 h (AST: 1854±974 UI/L, ALT: 713±392 UI/L, LDH: 2071±1524 UI/L, nonsignificant) and significantly lower after a 24-h reperfusion period (AST: 8458±2596 UI/L, P
