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Rat liver injury after normothermic ischemia is prevented by a phosphinic matrix metalloproteinase inhibitor ¹

RAFFAELE CURSIO², BERNARD MARI^*,23, KRYSTEL LOUIS^*, PHILIPPE ROSTAGNO, MARIE-CHRISTINE SAINT-PAUL, JEAN GIUDICELLI, VIRGINIE BOTTERO^*, PATRICK ANGLARD, ATHANASIOS YIOTAKIS^¶, VINCENT DIVE, JEAN GUGENHEIM and PATRICK AUBERGER^*

Laboratoire de Recherches Chirurgicales, IFR50,
^* INSERM U526, IFR50, Faculté de Médecine Pasteur,
Centre Antoine Lacassagne,
Service d’Anatomo-Pathologie, Hôpital Pasteur,
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; and
CEA, Département d’Ingénierie et d’Etudes des Protéines, CEA-Saclay, France

³Correspondence: INSERM U526, IFR50, Faculté de Médecine Pasteur, Nice, France. E-mail: bmari{at}unice.fr.

SPECIFIC AIMS

In this study, we have evaluated the expression of 9 matrix metalloproteinases (MMPs) and their endogenous inhibitors, the tissue inhibitors of MMPs (TIMPs) in a rat model of liver ischemia/reperfusion (I/R). While most of these genes are not constitutively expressed in the normal liver, they are induced in a specific time-dependent manner after I/R, suggesting that MMP/TIMP could play both deleterious and beneficial role after I/R. To investigate this possibility, we have tested the effect of a specific phosphinic MMP inhibitor on the acute liver I/R injury.

PRINCIPAL FINDINGS

1. Expression of MMP- and TIMP-specific transcripts after liver I/R
Using a model of partial liver I/R in rats, hepatic expression of 9 MMPs and 3 TIMPs was evaluated by Northern blot analysis (Fig. 1 ). In nonischemic liver lobes, expression of most MMPs and TIMPs was low or undetectable with the exception of gelatinase A and MT1-MMP. After I/R, MMP and TIMP expression was induced in a specific time-dependent pattern. The transcripts for collagenase 3 (MMP-13) and stromelysin 1 (MMP-3) were rapidly induced 3 h after reperfusion and returned to basal levels after 24–48 h. Gelatinase B (MMP-9) presented a similar profile but with a biphasic induction that was prolonged 96 h after I/R. Metalloelastase (MMP-12) and stromelysin 3 (MMP-11) were induced after 24–48 h and their expression remained elevated even after 1 wk. MT1-MMP (MMP-14) and gelatinase A (MMP-2) expression were indistinguishable with a low constitutive level and a peak 2–3 days after I/R followed by a slow decline. Finally, 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.

View larger version (49K): [in this window] [in a new window]   Figure 1. Expression of MMPs and TIMPs after I/R. Rats were subjected to an hepatic ischemia procedure. At different times after surgical procedure, the ischemic and normal liver lobes were collected and total RNAs and proteins were extracted. A) Northern blot analysis of MMPs and TIMPs. No detectable signal was obtained using MMP-7, MMP-10, and TIMP-3 probes. NI: nonischemic; I/R: ischemia/reperfusion. B) Zymography analysis of liver tissues.

2. Gelatinolytic activity in liver tissue after I/R
To verify that induction of MMP mRNAs correlated with an increased expression and activity of the corresponding proteins, we performed gelatin zymography (Fig. 1B ). This technique allows the detection of the two enzymatic activities 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, B ). The activity then decreased at 72 h, returning to basal level 1 wk after I/R. Active gelatinase B at 82 kDa was detectable only in ischemic conditions, indicating that the protein was induced and activated after I/R. In the case of gelatinase A, activity was poorly detected until 48 h; both the zymogen (72 kDa) and mature forms (62 kDa) increased after this time and remained stable during the course of reperfusion.

3. Administration of a MMP inhibitor decreases serum hepatic enzymes levels in I/R-treated rats
To evaluate the effect of MMP inhibition on ischemia-induced injury, rats were injected in the portal vein with 0.5 mg of the MMP inhibitor RXPO3 or vehicle alone (2% DMSO in PBS) twice two min prior to induction of ischemia and 2 min after clamp release. Blood samples were collected from animals 6 and 24 h later and serum levels of aspartate (AST), alanine (ALT) aminotranferases and lactate dehydrogenase (LDH) were measured (data not shown). The release of liver enzymes was slightly diminished in animals treated with RXPO3 after 6 h and significantly lower after a 24 h reperfusion period.

4. MMP inhibition decreases both necrosis and apoptosis in ischemic livers
The morphology of control or RXPO3-treated livers was then examined on paraffin-embedded sections. Histological analysis of an ischemic lobe 6 h after reperfusion showed large areas of necrosis and congestion with a heterogeneous localization, predominating in subcapsular and mediolobular areas. In RXPO3-treated group, both the surface and the intensity of liver necrosis were significantly reduced (data not shown). To evaluate the effect of MMP inhibition on apoptosis, TUNEL assay allowing detection of DNA fragmentation in situ was performed. Six hours after reperfusion, numerous hepatocytes from ischemic liver lobes were TUNEL-positive (Fig. 2 A, B), whereas virtually no TUNEL-positive hepatocytes were detected in the nonischemic liver lobes counterpart (Fig. 2C ). After 24 h, very large areas of TUNEL-positive cells were detected that could reach almost 50% of total hepatocytes (Fig. 2G, H ). In RXPO3-treated animals, the number of TUNEL positive cells decreased after 6 (Fig. 2A, B vs. D, E) and 24 h (Fig. 2G, H vs. I, J) of reperfusion. This inhibitory effect was confirmed by a quantitative analysis of hepatocyte apoptosis. Indeed, as shown in panel K, 18 ± 5% and 38 ± 11% of the cells were TUNEL positive in control rats vs. 9 ± 5% and 21 ± 9% in RXPO3-treated animals 6 h and 24 h after reperfusion, respectively.

View larger version (75K): [in this window] [in a new window]   Figure 2. Determination of liver apoptosis in situ by the TUNEL method. Terminal deoxyribonucleotidyl transferase D-UTP nick-end labeling method was carried out on 4 µm section prepared from nonischemic (C, F) or ischemic liver lobes (A, B, D, E, G–J) obtained from control (A–C, G, H) or RXPO3-treated rats (D–F, I, J) after a 6 (A–F) or 24 h (G–J) reperfusion period. K) Quantitative analysis of apoptotic hepatocytes in hepatic liver lobes from treated or untreated animals 6 and 24 h after I/R. The number of apoptotic cells observed in 2 different sections from 4 different rats was determined by cell counting on 8 different panels. Treatment decreased significantly the number of TUNEL-positive at 6 and 24 h compared with control group (*P<0.05, **P<0.02).

CONCLUSION AND SIGNIFICANCE

We show that ischemia/reperfusion of the liver is accompanied by profound modifications in MMP and TIMP expression. Most of these genes were induced sequentially but their overall induction could be classified into two different patterns. One group of genes is rapidly and transiently induced and includes stromelysin 1, gelatinase B, and collagenase 3. By contrast, the expression of gelatinase A, MT1-MMP, metalloelastase, stromelysin 3, TIMP-1, and -2 was delayed and prolonged, suggesting that these two sets of genes could play distinct functions after liver injury (Fig. 3 ). Gelatinase B is most widely found in inflammatory cells, and is likely to be expressed by Kupffer cells, the resident macrophages of the liver. Kupffer cells play a key role in liver injury induced by reperfusion, notably via a massive release of various cytokines such as tumor necrosis factor (TNF-). Two others MMPs, stromelysin 1 and collagenase 3, are also rapidly induced after I/R. These genes have been detected previously in both hepatocytes and nonparenchymal cells and their precise cellular localization in ischemic liver will also require further studies. Taken together, these data suggest that stromelysin 1, collagenase 3, and gelatinase B participate in the degradation of liver ECM after I/R and could play an important role during the inflammation phase (Fig. 3) .

View larger version (59K): [in this window] [in a new window]   Figure 3. Schematic diagram that summerizes the time course of MMP/TIMP expression after liver I/R.

Other MMP/TIMP members were induced later, suggesting a role for these proteins in repair or liver regeneration. Gelatinase A transcript showed a delayed up-regulation 24–48 h after I/R. The expression of MT1-MMP and TIMP-2, the components of the trimeric complex involved in gelatinase A activation, increased in parallel with gelatinase A levels (Fig. 1) . Another TIMP member, TIMP-1 was induced after I/R with a peak of expression at 48 h. Taken together, this expression profile may account for the diminished matrix degradation and the increased matrix deposition necessary to achieve reconstitution of the tissue. Finally, we also observe the up-regulation of two other MMPs in our model of normothermic liver I/R. Indeed, a strong elevation of metalloelastase, a macrophage-specific MMP was visible after 24 h of reperfusion, whereas stromelysin 3 presented a unique expression pattern with a delayed induction after 48 h of reperfusion followed by a stable expression.

Altogether, the complex MMP/TIMP expression patterns observed in our rat model of liver I/R strongly suggests that this family of protein play an important role in liver injury and/or repair. To investigate this hypothesis, rats were pre-treated with RXPO3, a new and potent phosphinic inhibitor of MMPs, before I/R procedure and its effect on early liver injury was evaluated. Administration of RXPO3 significantly reduced the level of transaminases and LDH. Moreover, reduced liver enzymes levels correlated with a significant inhibition of hepatocyte necrosis and apoptosis.

The mechanism by which ischemia/reperfusion leads to liver injury is presently not fully understood but is probably multifactorial. Release of oxygen-free radicals and inflammatory cytokines such as TNF- contribute to the destruction of liver cells after reperfusion. All these signals are known to induce apoptosis via both caspase-dependent and independent mechanisms but are also able to profoundly affect MMP/TIMP expression. Indeed, TNF- is a very potent inducer of gelatinase B in monocytes/macrophages and Kuppfer cells and is also able to induce the expression of several other MMPs in hepatic stellate cells. MMPs can alter cell behavior by their action on ECM and could also be involved in the migration of hepatic stellate cells and/or Kupffer cells which need to accumulate in the necrotic lesions. MMP inhibition would thus decrease inflammation and congestion resulting in diminished liver damage.

In conclusion, the present report provides the first study concerning the regulation of MMP/TIMP expression after liver I/R and demonstrates using a new selective inhibitor that MMP-mediated matrix breakdown participates in I/R liver injury. We therefore propose that MMP inhibitors may be of clinical relevance in liver-associated ischemic diseases or after liver transplantation.

ACKNOWLEDGMENTS

This work was supported by grants from The Institut National de la Santé et de la Recherche Médicale, University of Nice-Sophia Antipolis, The Ligue Nationale contre le Cancer and The Fondation de France. We thank Pr Jean Rosenbaum for helpful discussion and Anne Doye and Christian Petit for technical assistance.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0279fje; to cite this article, use FASEB J. (November 14, 2001) 10.1096/fj.01-0279fje

² R.C. and B.M. contributed equally to this work.

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