EARLY UPREGULATION OF MATRIX METALLOPROTEINASES FOLLOWING REPERFUSION TRIGGERS NEUROINFLAMMATORY MEDIATORS IN BRAIN ISCHEMIA IN RAT Diana Amantea,* Rossella Russo,* Micaela Gliozzi,y Vincenza Fratto,y Laura Berliocchi,*,z G. Bagetta,* G. Bernardi,} and M. Tiziana Corasanitiy,z *Department of Pharmacobiology, UCHAD Section of Neuropharmacology of Normal and Pathological Neuronal Plasticity, University of Calabria, 87036 Rende, Italy y Department of Pharmacobiological Sciences, Faculty of Pharmacy, University ‘‘Magna Graecia’’ of Catanzaro, 88100 Catanzaro, Italy z Mondino-Tor Vergata Center for Experimental Neuropharmacology, University of Rome Tor Vergata, 00133 Rome, Italy } CERC-Fondazione S. Lucia IRCCS, University of Rome Tor Vergata, 00133 Rome, Italy I. Introduction II. Methods A. Focal Cerebral Ischemia and Drug Treatments B. Neuropathology and Quantification of Ischemic Damage C. IL-1
ELISA D. Western Blotting E. In Situ Zymography F. Gel Zymography G. Fluorimetric Caspase-1 Activity Assay H. Statistical Analysis III. Results IV. Discussion References Abnormal expression of matrix metalloproteinases (MMPs) has been implicated in the pathophysiology of neuroinflammatory processes that accompany most central nervous system disease. In particular, early upregulation of the gelatinases MMP-2 and MMP-9 has been shown to contribute to disruption of the blood–brain barrier and to death of neurons in ischemic stroke. In situ zymography reveals a significant increase in gelatinolytic MMPs activity in the ischemic brain hemisphere after 2-h middle cerebral artery occlusion (MCAo) followed by 2-h reperfusion in rat. Accordingly, gel zymography demonstrates that expression and activity of MMP-2 and MMP-9 are enhanced in cortex and striatum ipsilateral to the ischemic insult. The latter eVect appears to be instrumental for development INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 82 DOI: 10.1016/S0074-7742(07)82008-3 149 Copyright 2007, Elsevier Inc. All rights reserved. 0074-7742/07 $35.00 150 AMANTEA et al. of delayed brain damage since administration of a broad spectrum, highly specific MMPs inhibitor, GM6001, but not by its negative control, results in a significant (50%) reduction in ischemic brain volume. Increased gelatinase activity in the ischemic cortex coincides with elevation (166% vs sham) of mature interleukin-1
(IL-1
) after 2-h reperfusion and this does not appear to implicate a caspase-1dependent processing of pro(31 kDa)-IL-1
to yield mature (17 kDa) IL-1
. More importantly, when administered at a neuroprotective dose GM6001 abolishes the early IL-1
increase in the ischemic cortex and reduces the cleavage of the cytokine proform supporting the deduction that MMPs may initiate IL-1
processing. In conclusion, development of tissue damage that follows transient ischemia implicates a crucial interplay between MMPs and mediators of neuroinflammation (e.g., IL-1
), and this further underscores the therapeutic potential of MMPs inhibitors in the treatment of stroke. I. Introduction Interleukin-1
(IL-1
) is a proinflammatory cytokine that has been identified as an important mediator of neurodegeneration induced by excitatory or traumatic brain injury and, most notably, by experimental cerebral ischemia in rodents (Rothwell, 2003). Induction of IL-1
mRNA has been shown in rats following either permanent (Buttini et al., 1994; Liu et al., 1993) or transient (Wang et al., 1994) middle cerebral artery occlusion (MCAo). Accordingly, IL-1
protein increases very early following permanent MCAo (Davies et al., 1999; Legos et al., 2000) and peaks within hours of reperfusion in transient focal ischemic models in rodents (Hara et al., 1997b; Zhang et al., 1998). Intracerebral injection of neutralizing anti-IL-1
antibody to rats reduces ischemic brain damage (Yamasaki et al., 1995) and both intracerebroventricular and systemic administration of IL-1receptor antagonist (IL-1ra) markedly reduce brain damage induced by focal stroke, further implicating IL-1
in ischemic pathophysiology (Garcia et al., 1995; Mulcahy et al., 2003; Relton and Rothwell, 1992; Relton et al., 1996). IL-1
is synthesized as a precursor molecule, pro-IL-1
, which is cleaved and converted into the mature, biologically active, form of the cytokine by caspase-1, formerly referred to as interleukin-1
converting enzyme (ICE; Black et al., 1988; Howard et al., 1991; Thornberry et al., 1992). Inhibition of caspase-1 by Ac-YVAD-cmk aVords neuroprotection in rodent models of permanent (RabuVetti et al., 2000) or transient (Hara et al., 1997b) MCAo (tMCAo), and evidence from knockout mice indicates that caspase-1 is important in the development of cerebral ischemic damage (Friedlander and Yuan, 1998; Schielke et al., 1998). MMPS MODULATE IL-1
PRODUCTION IN BRAIN ISCHEMIA 151 However, to date, it is not clear whether neuroprotection yielded by caspase-1 preferring inhibitors is mediated by reduced IL-1
production or by interference with the death process. Although in vitro studies have clearly established the role of ICE in the maturation of IL-1
, evidence from ICE-deficient mice suggests that cytokine activation might also involve other mechanisms (Fantuzzi et al., 1997). Matrix metalloproteinases (MMPs) have been suggested to contribute to the biological processing of IL-1
as they have been shown to be involved in both the maturation and inactivation of the cytokine in vitro (Ito et al., 1996; Schönbeck et al., 1998). MMPs are zinc-dependent endopeptidases, classically recognized as matrixdegrading enzymes implicated in tissue remodeling during development, wound healing, and angiogenesis. MMPs are expressed as zymogens that are activated on disruption of the zinc–thiol interaction between the catalytic site and the prodomain. MMPs cleave protein components of the extracellular matrix (ECM) such as collagen, proteoglycan, and laminin, but also process a number of cell surface and soluble proteins including receptors, cytokines, and chemokines (Sternlicht and Werb, 2001). In addition to their physiological roles, MMPs are markedly upregulated in the central nervous system (CNS) in response to injury and have been implicated in the propagation and regulation of neuroinflammatory processes that accompany most CNS disease (Cunningham et al., 2005; Rosenberg, 2002). Evidence suggests that abnormal MMP activity plays a role in the pathophysiology of cerebral ischemia. In particular, the gelatinases MMP-2 and MMP-9 become activated following focal brain ischemia and participate to the disruption of the blood–brain barrier (BBB) and hemorrhagic transformation following injury both in animal models (Asahi et al., 2000; Heo et al., 1999; Romanic et al., 1998; Rosenberg et al., 1998) and stroke patients (Horstmann et al., 2003; Rosell et al., 2006). Treatment with MMP inhibitors or MMP neutralizing antibodies has been shown to decrease infarct volume and prevent BBB disruption after permanent and tMCAo in rodents (Asahi et al., 2000; Gasche et al., 2001; Romanic et al., 1998). MMP-9, but not MMP-2 (Asahi et al., 2001a), gene knockout is associated with reduced infarct size and less BBB damage in mouse models of ischemic stroke (Asahi et al., 2000, 2001b). This large body of evidence suggests that MMPs and, most notably, the gelatinases MMP-2 and MMP-9 might contribute to the development of ischemic brain damage, though the underlying mechanisms need to be discovered. Various studies have emphasized the role of MMPs and their endogenous inhibitors (TIMPs) in the regulation of neuronal cell death through the modulation of excitotoxicity (Jourquin et al., 2003), anoikis (Gu et al., 2002), calpain activity (Copin et al., 2005), death receptor activation (Wetzel et al., 2003), neurotrophic factor bioavailability (Lee et al., 2001), and production of neurotoxic products (Gu et al., 2002; Zhang et al., 2003a). This suggests that, in addition to ECM degradation, MMPs might elicit some direct, pathogenic eVects, which contribute to brain tissue damage under various neuropathological conditions. 152 AMANTEA et al. Here, we suggest that cytokine maturation may represent a likely mechanism by which MMPs contribute to ischemic neuronal death and postulate that MMPs might be involved in the early increase of IL-1
occurring following tMCAo in rat. In fact, increased gelatinolytic, namely, MMP-2 and MMP-9, activity is observed as early as 2 h following reperfusion, and this is coincident with a significant increase of IL-1
levels in the ischemic cortex. More interestingly, pharmacological inhibition of gelatinolytic activity prevents elevation of cortical IL-1
levels and results in significant neuroprotection. II. Methods A. FOCAL CEREBRAL ISCHEMIA AND DRUG TREATMENTS Adult male Wistar rats (Charles River, Calco, Como, Italy) were housed under controlled environmental conditions with ambient temperature of 22  C, relative humidity of 65%, and 12-h light:12-h dark cycle, with free access to food and water. Brain ischemia was induced by MCAo in rats weighing 280–320 g by intraluminal filament, using the relatively noninvasive technique previously described by Longa et al. (1989). Briefly, rats were anesthetized with 5% isoflurane in air and were maintained with the lowest acceptable concentration of the anesthetic (1.5–2%). Body temperature was measured with a rectal probe and was kept at 37  C during the surgical procedure with a heating pad. Under an operating microscope, the external and internal right carotid arteries were exposed through a neck incision. The external carotid artery was cut approximately 3 mm above the common carotid artery (CCA) bifurcation and a silk suture was tied loosely around the external carotid stump. A silicone-coated nylon filament (diameter, 0.28 mm) was then inserted into the external carotid artery and gently advanced into the internal carotid artery, approximately 18 mm from the carotid bifurcation, until mild resistance was felt, thereby indicating occlusion of the origin of the middle cerebral artery in the Willis circle. The silk suture was tightened around the intraluminal filament to prevent bleeding. The wound was then sutured and anesthesia discontinued. Sham rats were exposed to the same surgical procedure without occlusion of MCA. To allow reperfusion, rats were briefly reanesthetized with isoflurane, and the nylon filament was withdrawn 2 h after MCAo. After the discontinuation of isoflurane and wound closure, the animals were allowed to awake and were kept in their cages with free access to food and water. N-[(2R)-2-(Hydroxamidocarbonylmethyl)-4-methylpenthanoyl]-L-tryptophan methylamide (GM6001; also known as Galardin), a potent broad-range inhibitor of MMPs (Levy et al., 1998), and its negative control (N-t-butoxycarbonyl-L-leucyl-Ltryptophan methylamide, GM6001 negative control), obtained from Calbiochem (La Jolla, CA), were dissolved in DMSO and administered in a volume of 2 l through the external carotid artery (i.a.), 15 min prior to MCAo. MMPS MODULATE IL-1
PRODUCTION IN BRAIN ISCHEMIA 153 All the experimental procedures were in accordance to the guidelines of the European Community Council Directive 86/609, included in the D.M. 116/ 1992 of the Italian Ministry of Health. B. NEUROPATHOLOGY AND QUANTIFICATION OF ISCHEMIC DAMAGE Cerebral infarct volume was evaluated 22 h after reperfusion in rats subjected to 2-h MCAo. Rats were sacrificed by decapitation and the brains were rapidly removed. Eight serial sections from each brain were cut at 2-mm intervals from the frontal pole using a rat brain matrix (Harvard Apparatus, Massachusetts). To measure ischemic damage, brain slices were stained in a solution containing 2% 2,3,5-triphenyltetrazolium chloride (TTC) in saline, at 37  C. After 10-min incubation, the slices were transferred to 10% neutral buVered formaldehyde and stored at 4  C prior to analysis. Images of TTC-stained sections were captured using a digital scanner and analyzed using an image analysis software (ImageJ, version 1.30). The infarct volume (mm3) was calculated by summing the infarcted area (unstained) of the eight sections and multiplying by the interval thickness between sections as previously described (Li et al., 2000). C. IL-1
ELISA Immunoreactive IL-1
levels were analyzed in individual brain cortical tissue homogenates by an established, rat-specific, sandwich ELISA previously described (Corasaniti et al., 2001; Hagan et al.,1996), using an immunoaYnity-purified polyclonal sheep anti-rat IL-1
-coating antibody (1 g/ml) and a biotinylated, immunoaYnity-purified polyclonal sheep anti-rat IL-1
-detecting antibody (1:1000 dilution), kindly provided by Dr. Stephen Poole (National Institute of Biological Standards and Controls, NIBSC, Hertfordshire, United Kingdom). Poly-horseradish peroxidase-conjugated streptavidin (CLB, Amsterdam, the Netherlands) was used at 1:5000 dilution and the color was developed by using the chromogen o-phenylenediamine. Optical densities (OD) were read at 492 nm by using an automated plate reader (Multiscan MS, Labsystems, Helsinki, Finland) and cytokine levels were calculated by interpolation from a standard curve obtained from recombinant rat IL-1
(0.0–1000 pg/ml). Data were corrected for protein concentration and the results expressed as picogram of IL-1
per milligram of protein. D. WESTERN BLOTTING For Western blotting analysis of mature and pro-IL-1
immunoreactivities, 20 g of proteins, from the same aliquots used for ELISA assay, were resolved by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 154 AMANTEA et al. transferred to nitrocellulose membranes (Optitran BA-S 83, Schleicher & Schuell Bioscience, Dassel, Germany), and probed overnight at 4  C with a polyclonal sheep anti-rat antibody, which recognizes both the precursor and mature forms of this cytokine (S1002/BM, NIBSC; 7.5 g/ml). The membranes were then incubated with horseradish peroxidase-conjugated anti-sheep IgG (1:5000 dilution; Chemicon International, Inc., Temecula, CA) for 1 h at room temperature. Immunoreactivity was visualized by chemiluminescent detection (Amersham Biosciences, GE Healthcare, Milan, Italy). E. IN SITU ZYMOGRAPHY In situ zymography with the MMP fluorogenic substrate DQ-gelatin-FITC (Molecular Probes, Eugene, OR) was performed on OCT-embedded fresh brain cryostat-cut sections obtained from rat sacrificed after 2-h MCAo followed by 2-h reperfusion, as previously described (Gu et al., 2002). Briefly, rat brains were dissected out, immediately embedded in OCT (Tissue-Tek, Pennsylvania) and frozen on dry ice; 15-m-thick coronal sections were cut using a cryostat, air-dried for 1 h at room temperature, then rehydrated in PBS, and incubated overnight at 37  C with the quenched fluorogenic substrate DQ-gelatin-FITC (40 g/ml in PBS). The excess of fluorogenic substrate was washed out by three washing steps, 5 min each, in PBS. Sections were fixed with 2% paraformaldehyde in PBS for 5 min and nuclei counterstained with propidium iodide (0.5 g/ml) for 20 min at room temperature. Mounted slides were examined by confocal microscopy to detect the green fluorescence due to gelatinolytic activity. F. GEL ZYMOGRAPHY MMP-2 (gelatinase A) and MMP-9 (gelatinase B) gelatinolytic activities were detected by gelatin gel zymography (Gu et al., 2005). Individual brain striatal, cortical, and hippocampal tissue samples (n ¼ 3 per experimental group) were homogenized in ice-cold Tris-buVered saline (TBS), containing 150-mM NaCl, 5-mM CaCl2, 0.05% Brij35, pH 7.6, 0.02% NaN3, 1% Triton X-100, 100-M PMSF, and a protease inhibitor cocktail (Sigma, Milan), and centrifuged at 14,000  g for 20 min at 4  C. Supernatants were subjected to aYnity precipitation with gelatin-conjugated Sepharose beads (Gelatine-Sepharose 4B, Amersham Biosciences, GE Healthcare, Milan, Italy), overnight at 4  C. The bound proteins were eluted from the beads in TBS containing 10% DMSO by shaking for 1 h at 4  C. Ten microliters of extracted brain samples were diluted (1:1) in a nonreducing loading buVer (0.0625-M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.25% bromophenol blue) and subjected to electrophoresis through a 10% SDS MMPS MODULATE IL-1
PRODUCTION IN BRAIN ISCHEMIA 155 polyacrylamide gel copolymerized with 0.1% gelatin. After electrophoretic separation, the gel was incubated (2  30 min) in 2.5% Triton X-100 to remove SDS, washed two times, 15 min each, with water and then incubated for 40 h at 37  C in a developing buVer containing 50-mM Tris–HCl, pH 7.78; 10-mM CaCl2 and 0.02% NaN3. Following incubation, the gel was stained for 1 h with 0.25% Coomassie Brilliant Blue R-250 diluted with methanol (50%) and acetic acid (10%) and finally destained in a solution of acetic acid:methanol:water (1:3:6). Enzyme activity attributed to MMP-2 and MMP-9 was visualized (on the basis of molecular weight) in the gelatin-containing zymograms as clear bands against a blue background. G. FLUORIMETRIC CASPASE-1 ACTIVITY ASSAY Individual brain cortical tissue samples (n ¼ 4 per experimental group) were rapidly dissected out and homogenized in ice-cold lysis buVer (50-mM HEPES, pH 7.4, 150-mM NaCl, 5-mM MgCl2, 5-mM EDTA, 0.1% CHAPS, 5-mM DTT, 10 g/ml pepstatin A, 10-g/ml leupeptin, 10-g/ml aprotinin); following 10 min incubation on ice, samples were centrifuged at 12,000  g for 10 min at 4  C and protein concentration in supernatants was determined by the DC protein assay (Bio-Rad Laboratories, Milan, Italy). Brain cortical supernatants were diluted in assay buVer (100-mM HEPES, pH 7.4, 5-mM EDTA, 0.1% CHAPS, 5-mM dithiothreitol, 10% glycerol) to a final concentration of 1.2 g protein/l and incubated in triplicate in a 96-well clear-bottom plate with the fluorogenic substrate acetyl-Trp-Glu-His-Asp-7-amino-4-methylcoumarin (Ac-WEHD-AMC; 10 M; Bachem) (Rano et al., 1997). Production of fluorescent-free AMC, released by caspase-1 activity, was monitored over 60 min at 37  C using a microplate fluorometer (Victor2 multilabel counter, PerkinElmer Life Sciences; excitation, 355 nm; emission, 460 nm). Specific contribution of caspase-1 activity in each brain extract was determined by preincubating parallel sample aliquots with the caspase-1 preferring inhibitor acetyl-Trp-GluHis-Asp-aldehyde (Ac-WEHD-CHO; 10 M; Bachem) (Garcia-Calvo et al., 1998) for 10 min at 37  C prior to the addition of the caspase substrate; the diVerence between the substrate cleavage activity in the absence and presence of Ac-WEHD-CHO was regarded as specific caspase-1 activity. The increase in fluorescence was linear for 40 min after addition of the fluorogenic substrate. Data were analyzed by linear regression within the linear range of the enzymatic reaction and the fluorescence units were converted to micromoles of AMC by using a standard curve generated with free AMC (Calbiochem); the results were expressed as micromoles of free AMC released per minute per milligram of protein and reported as mean  SEM. 156 AMANTEA et al. H. STATISTICAL ANALYSIS Data are expressed as mean  SEM and statistical analysis performed by ANOVA followed by Dunnett’s or Tukey’s post hoc tests using the Prism 3 program (GraphPAD Software for Science, San Diego, CA). DiVerences were considered statistically significant when p < 0.05. III. Results In situ zymography revealed a considerable increase of total gelatinolytic MMP activity in the brain of rats subjected to 2-h MCAo followed by 2-h reperfusion (Fig. 1). This early increase in gelatinolytic activity was evident in the brain areas supplied by the MCA, including the striatum (E and G) and the periinfarct motor cortex (M and O) as compared to the corresponding regions of sham-operated animals (A, C, I, and K). MMPs broadly colocalized with nuclear DNA staining as shown by the merging of the in situ zymography signal with propidium iodide staining (D, H, L, and P). Consistent with these findings, gel zymography assay demonstrated that both MMP-2 and MMP-9 levels were increased in the ipsilateral, ischemic cortex of rats subjected to tMCAo, as compared to the contralateral, nonischemic hemisphere; whereas no diVerence was found in the cortical samples from shamoperated animals (Fig. 2A). As shown in Fig. 2B, enhanced gelatinolytic activity corresponding to MMP-2 and MMP-9 was also observed in the striatum with less significant changes in the hippocampus, a brain region only marginally aVected by the ischemic insult produced by MCAo (Fig. 2B). On the basis of molecular weight, MMP-9 increases predominantly in the dimeric (250-kDa band) and latent (90- to 95-kDa band) ‘‘pro’’ forms, whereas enhanced levels of MMP-2 following ischemia/reperfusion occurred mainly in the active forms (65- to 67-kDa band). The levels of MMP-9 were low, in comparison to MMP-2, in both ischemic and nonischemic hemispheres and throughout the brain regions examined, as shown by the weaker gelatinolytic bands on the zymography gels. To determine whether administration of the broad spectrum MMP inhibitor, GM6001, aVects brain gelatinolytic activity following brain ischemia, we performed gel zymography on striatal tissue samples from rats exposed to 2-h MCAo followed by 2-h reperfusion. As shown in Fig. 3, intra-arterial administration of GM6001 (5 g/rat) prevented the increase of MMP-2 and MMP-9 activity in the striatum ipsilateral to the ischemic insult, otherwise observed after pretreatment with the GM6001 negative, inactive, control. Inhibition of MMPs by GM6001 resulted in a significant reduction of MCAoinduced brain damage as revealed 22 h after reperfusion by the TTC-staining 157 MMPS MODULATE IL-1
PRODUCTION IN BRAIN ISCHEMIA In situ zymography B C Merged D Striatum Sham A In situ zymography PI staining F G 97 mm H MCAo E 97 mm 97 mm 300 mm 101.37 mm 300 mm I 101.37 mm K L Cortex Sham J 101.37 mm 300 mm M 99.32 mm 99.32 mm O P MCAo N 99.32 mm 300 mm 98 mm 98 mm 98 mm FIG. 1. Increased MMP gelatinolytic activity after transient focal cerebral ischemia. Typical in situ zymography with the MMP fluorogenic substrate DQ-gelatin FITC showing the robust increase of MMP gelatinolytic activity in the striatum (E and G) and cortex (M and O) after 2-h occlusion of MCA followed by 2-h reperfusion compared with sham-operated control (A, C, I, and K). Activity appeared as a green fluorescent product and developed after incubation of coronal sections (10-m in thickness) with the fluorogenic substrate DQ-gelatin FITC (panels A, C, E, G, I, K, M, and O). In situ zymography merged with nuclear DNA staining with propidium iodide (PI) dye (red plus green in panels D, H, L, and P) shows that increased gelatinolytic activity (green fluorescence in panels G and O) broadly colocalizes with DNA staining (red fluorescence in panels F and N) in ischemic striatum (H) and cortex (P). Scale bar: 97–101 m in B–D, F, G, H, J, K, L, N, O, and P; 300 m in A, E, I, and M. technique. As illustrated in Fig. 4, administration of GM6001 negative control (GM Neg, 5 g/rat, i.a.), given 15 min before tMCAo, resulted in an extensive brain damage, comparable to vehicle-treated control (data not shown), involving the cortex and the striatum (infarct volume: 502.6  18.4 mm3); this was significantly reduced by administration of 5 g of GM6001 (infarct volume: 158 AMANTEA et al. A B Sham C Striat tMCAo I C C I Hipp I C I MMP-9 dimer MMP-9 dimer Pro-MMP-9 MMP-9 Pro-MMP-9 MMP-9 MMP-2 MMP-2 FIG. 2. Increased MMP-9 and MMP-2 after transient focal cerebral ischemia. (A) Typical gel zymography of brain cortical homogenates, subjected to aYnity precipitation with gelatin-conjugated Sepharose beads, after 2-h occlusion of MCA followed by 2-h reperfusion. Representative zymogram gel showing elevation of MMP-9 and MMP-2 in the ipsilateral (I), ischemic side as compared to contralateral (C), nonischemic side in a rat subjected to tMCAo but not in a sham-operated animal. (B) Typical gel zymography of striatal and hippocampal brain homogenates, subjected to aYnity precipitation with gelatin-conjugated Sepharose beads, after 2-h transient cerebral ischemia and 2-h reperfusion. Representative zymogram gel showing elevation of MMP-9 and MMP-2 in the ipsilateral (I), ischemic side as compared to contralateral (C), nonischemic side. GM Neg C I GM6001 C I MMP-9 dimer Pro-MMP-9 MMP-9 MMP-2 FIG. 3. GM6001 decreases MMP-9 and MMP-2 activity in the brain of rats subjected to transient focal cerebral ischemia. Rats pretreated with the broad-spectrum metalloproteinase inhibitor, GM6001 (5 g/rat i.a., 15 min before MCAo), but not with the inactive, negative control of GM6001 (GM Neg; 5 g/rat i.a.), show decreased MMP-9 and MMP-2 activity after ischemia (2-h MCAo followed by 2-h reperfusion) as assessed by gel zymography of brain striatal homogenates subjected to aYnity precipitation with gelatin-conjugated Sepharose beads. 159 MMPS MODULATE IL-1
PRODUCTION IN BRAIN ISCHEMIA A GM Neg GM6001 C B Infract volume (mm3) Infract area (mm2) 75 50 25 0 600 400 *** 200 0 0 1 2 3 4 5 6 Coronal slice 7 8 GM Neg GM6001 (0.05 mg) GM6001 (5 mg) FIG. 4. GM6001 protects against brain damage after transient focal cerebral ischemia. (A) Histological evidence for GM6001-mediated neuroprotection against brain damage produced by tMCAo. After 2-h MCA and 22-h reperfusion, eight consecutive coronal sections from each brain were cut at 2-mm intervals from the frontal pole and incubated in TTC, which stains viable tissue red but not infracted areas. GM6001 or its negative control (GM Neg) were dissolved in DMSO and administered (5 g/rat, 2-l injection volume) into the external carotid artery 15 min before MCAo. Quantification of infarct area (B) and volume (C) by TTC staining revealed significant neuroprotection by GM6001 (5 g/rat) as compared to its negative control. Infarct volumes were determined after 2h MCAo followed by 22-h reperfusion by summing the infarcted area of the eight TTC-stained sections and multiplying by the interval thickness between sections. Values are expressed as mean  SEM and analyzed by ANOVA followed by Dunnett’s post hoc test (n ¼ 3 rats per experimental group). *** denotes p < 0.001 versus GM Neg. 251.9  9.5 mm3, p < 0.001 vs GM Neg), whereas a lower dose (0.05 g) resulted ineVective (infarct volume: 467.0  3.9 mm3). Under our present experimental conditions, ELISA assay of IL-1
revealed significantly increased cytokine levels in the ipsilateral cortex of rats subjected to MCAo followed by 2-h reperfusion (Fig. 5A), whereas a less marked increase was detected following 1-h reperfusion (data not shown). More importantly, cytokine 160 AMANTEA et al. Contralateral Ipsilateral A # ** IL-1b (pg/mg protein) 12.5 10.0 7.5 5.0 2.5 0.0 Sham Caspase-1 activity (mmol AMC/min/mg protein) B tMCAo Contralateral Ipsilateral 0.20 0.15 0.10 0.05 0.00 Sham tMCAo FIG. 5. Transient focal cerebral ischemia increases IL-1
levels but not caspase-1 activity in the ischemic cortex of rat. (A) Immunoreactive IL-1
levels are increased in the ipsilateral (I), ischemic cortex of rats (n ¼ 6) subjected to 2-h occlusion of MCA followed by 2-h reperfusion as compared to sham-operated animals (n ¼ 4). Immunoreactive IL-1
levels were assayed in individual brain cortical tissue samples by an established, rat specific, sandwich ELISA. IL-1
levels were corrected for protein concentration and the results expressed as pg of IL-1
per milligram of protein. Data are expressed as mean  SEM (n ¼ 4–6 per group). The resulting means were evaluated statistically for diVerences using ANOVA followed by Tukey-Kramer test for multiple comparisons. # denotes p < 0.05 versus control, contralateral cortex (C); ** denote p < 0.01 versus sham (contralateral and ipsilateral). (B) Caspase-1 activity does not increase in the ischemic cortex of rats subjected to transient occlusion (2 h) of MCA followed by 2-h reperfusion. Caspase-1 activity was determined by measuring cleavage of the fluorogenic substrate Ac-WEHD-AMC in individual cortical homogenates obtained from shamoperated and ischemic rats (n ¼ 4 per group). Data are expressed as micromoles of free AMC released per minute per milligram of protein and reported as mean  SEM. production was not associated with enhanced caspase-1 activity as determined by measuring cleavage of the fluorogenic substrate Ac-WEHD-AMC (Fig. 5B). To evaluate whether the observed elevation MMPs activity could contribute to IL-1
processing and maturation, we used a pharmacological approach. After MMPS MODULATE IL-1
PRODUCTION IN BRAIN ISCHEMIA 161 pretreatment with the broad spectrum MMPs inhibitor, GM6001, IL-1
levels measured in the cortex ipsilateral to the ischemic insult (2-h MCAo followed by 2-h reperfusion) were comparable with levels detected in the contralateral nonischemic brain tissue. By contrast, administration of GM6001 negative control did not aVect the increase of IL-1
observed in the cortex ipsilateral to the ischemic insult (Fig. 6A). Western blotting analysis confirmed that mature (17-kDa band) IL-1
levels were significantly reduced in the ischemic cortex from rats treated with GM6001 as compared to GM6001 negative control (Fig. 6B). By contrast, pro-IL1
(33- to 31-kDa band) levels were not aVected by GM6001 treatment. Moreover, we detected a strong band (about 28 kDa) corresponding to a cleavage product of the 33- to 31-kDa pro-IL-1
which was less intense in the cortex of rats treated with GM6001 as compared to GM negative control (Fig. 6B). IV. Discussion Despite the numerous reports implicating IL-1
in the neurodegeneration associated with ischemic insult, the role of caspase-1 in cytokine maturation following injury is still controversial. Here we demonstrate that IL-1
increases very early following tMCAo and this appears to occur independently from caspase-1 activation. Interestingly, we found that inhibition of MMPs activity prevented cytokine enhancement in the ischemic cortex, thus suggesting that MMPs might contribute to the early IL-1
maturation following a transient ischemic insult. The role of MMPs in the pathophysiology of ischemic stroke has been widely investigated particularly regarding their ability to degrade the neurovascular matrix, thus leading to BBB damage, hemorrhage, and anoikis-like cell death triggered by disruption of cell-matrix homeostasis (see Cunningham et al., 2005; Rosenberg, 2002). An early and progressive increase of MMP-9 expression has been detected particularly in endothelial cells in the ischemic hemisphere following tMCAo (Planas et al., 2001; Zhao et al., 2006) and has been implicated in the early stages of tissue injury produced by the ischemic insult (Romanic et al., 1998). Although the involvement of MMP-2 during the early stages after stroke has been questioned (Asahi et al., 2001a), there is evidence for a transient enhancement of MMP-2 early after temporary ischemia, which has been suggested to contribute to the eVects of reperfusion and to the early opening of the BBB (Planas et al., 2001; Rosenberg et al., 1998). At later stages following tMCAo, a redistribution of gelatinases in the neurovascular unit does occur, with a massive increase of MMP-2 expression in microglia/macrophages (Planas et al., 2001; Rosenberg et al., 2001) and MMP-9 in astrocytes and neurons (Zhao et al., 2006). The former eVect has been suggested to facilitate migration of macrophages into the ischemic lesion, whereas the latter might represent an endogenous mechanism involved in 162 AMANTEA et al. A B GM Neg Contralateral Ipsilateral IL-1b (pg/mg protein) 10.0 * 7.5 GM6001 Std 33 kDa 31 kDa 28 kDa 17 kDa 5.0 2.5 28 kDa 0.0 GM Neg GM6001 17 kDa b-actin FIG. 6. GM6001 abrogates the increase of cortical IL-1
induced by tMCAo and reduces the expression of pro-IL-1
cleavage products. (A) GM6001 (5 g/rat i.a.), but not the inactive form of the metalloproteinase inhibitor, GM6001 negative control (GM Neg; 5 g/rat i.a.), abrogates increase of cortical IL-1
levels in the ischemic cortex of rats undergone tMCAo (2h/2h reperfusion). Immunoreactive IL-1
levels were assayed in individual brain cortical tissue samples (n ¼ 3 per group) by an established, rat specific, sandwich ELISA. IL-1
levels were corrected for protein concentration and the results expressed as picograms of IL-1
per milligram of protein. Data are expressed as mean  SEM values (n ¼ 3 per group). The resulting means were evaluated statistically for diVerences using ANOVA followed by Tukey-Kramer test for multiple comparisons. * denotes p < 0.05 versus GM6001. (B) Pro-IL-1
immunoreactivity of ipsilateral, ischemic brain cortical homogenates obtained from rats subjected to tMCAo (2 h plus 2 h reperfusion) and pretreated with GM6001 (5 g/rat i.a.) or with its negative control (GM Neg; 5 g/rat i.a.) was assessed by Western blotting analysis using an antibody specific for pro-IL-1
. The antibody detected a strong band of about 28 kDa which has been previously described as a cleavage product of the 33- to 31-kDa pro-IL-1
, together with less intense bands of 33 and 31 kDa and a band of 17 kDa corresponding to mature IL-1
as determined by the use of recombinant mouse IL-1
employed as a standard (Std). As compared to animals pretreated with the inactive form of GM6001, a reduction of the intensity of IL-1
is evident in the cortex of GM6001-treated animals (upper panel in B) and this is associated to a reduction of the intensity of the 28-kDa cleavage product band as better shown in middle panel in B which represents the same gel as in upper panel but at a lower exposure time. the resolution phase. In fact, inhibition of MMP-9 between 7 and 14 days after stroke has been reported to result in a substantial reduction in the number of neurons and new vessels implicated in neurovascular remodeling (Zhao et al., 2006). These findings underscore the complexity of MMPs activity during tissue injury, ranging from detrimental eVects during the early phases after stroke to beneficial roles at later stages (Yong, 2005). MMPS MODULATE IL-1
PRODUCTION IN BRAIN ISCHEMIA 163 Here we demonstrate that increased gelatinolytic activity is detected in situ as early as 2 h following reperfusion both in the cortex and striatum of rats exposed to 2-h MCAo. This is confirmed by gel zymography revealing that active MMP-2, as well as pro- and dimeric forms of MMP-9 are significantly increased in the brain regions aVected by the ischemic insult. Previous studies have demonstrated that latent MMP-9 (dimer and propeptide forms) levels increase after focal (Asahi et al., 2000; Gu et al., 2002, 2005; Romanic et al., 1998; Rosenberg et al., 1996 ) and global cerebral ischemia in rodents (Magnoni et al., 2004; Rivera et al., 2002). Accordingly, here we observed a gelatinolytic band around 250 kDa, which increases significantly following brain ischemia/reperfusion. Similar gelatinolytic activity (200- to 250-kDa band) was reported in the hippocampus of rats subjected to global brain ischemia (Rivera et al., 2002) and in the brain of mice treated with LPS (Pagenstecher et al., 2000). These high-molecular-weight bands may correspond to homodimers produced intracellularly during the maturation process of the enzyme or to complexes of dimerized MMP-9, favored by increases in the gelatinase B/TIMP-1 ratio (Dubois et al., 1998; Goldberg et al., 1992), or to complexes of gelatinase B and integrins (Brooks et al., 1996). It is widely accepted that uncontrolled expression of MMPs may result in tissue damage and inflammation, though the mechanisms involved are still poorly understood, especially concerning those leading to neuronal cell death. Activated MMP-9 has been demonstrated to directly induce neuronal apoptosis both in vitro and in vivo after focal cerebral ischemia/reperfusion (Gu et al., 2002). Some studies have suggested that MMP-9 contributes to neuronal cell death via proteolysis of basement membrane proteins, including laminin (Asahi et al., 2001b; Castellanos et al., 2003; Chen et al., 2003; Gu et al., 2005; Horstmann et al., 2003). MMPs are also related to activation and release of several bioactive molecules such as cytokines, chemokines, and growth factors (see Parks et al., 2004). Here we suggest that brain tissue damage produced by MMPs in the ischemic brain might be linked to cleavage and activation of neuroinflammatory mediators. In fact, increased gelatinolytic activity is associated with a rise of mature IL-1
production in the ischemic cortex and, most importantly, inhibition of MMPs activity by GM6001 prevents the early increase of IL-1
yielded by the ischemic insult in the cortex and this results in significant neuroprotection. There is in vitro evidence that MMPs activate pro-IL-1
proteolitically and cleave the activated form of IL-1
to an inactive form, thereby providing both positive and negative regulation (Ito et al., 1996; Schönbeck et al., 1998). Our results extend these findings and suggest that MMPs contribute to maturation of IL-1
also in vivo, under pathophysiological conditions such as brain ischemia, by a mechanism independent from caspase-1 activation. Increased IL-1
generated via mechanisms other than caspase-1 has also been reported in the brain of rats exposed to kainic acid or 3,4-methylenedioxymethamphetamine (Eriksson et al., 1999; O’Shea et al., 2005) and in an animal model of neuroAIDS (Corasaniti et al., 2005), 164 AMANTEA et al. further emphasizing that, under certain circumstances, cytokine activation might involve proteases other than caspase-1 (Fantuzzi et al., 1997). Although ICE has been strongly implicated in the development of ischemic brain damage (Hara et al., 1997a,b; Schielke et al., 1998; Yang et al., 1999), we failed to detect increase in caspase-1 activity during the early stages after reperfusion. In animal models of MCAo, there is evidence that caspase-1 activity is enhanced shortly after the induction of ischemia (Benchoua et al., 2001) and ICElike protease activity increases few hours after reperfusion (Hara et al., 1997a). Nevertheless, some studies have revealed that caspase-1 displays a biphasic activation after the ischemic insult (Benchoua et al., 2001), whereas others have failed to detect any change in the expression of ICE after permanent MCAo (Asahi et al., 1997). Therefore, we cannot exclude that under our experimental conditions caspase-1 may be activated at some stage following tMCAo, thus contributing to tissue damage through mechanisms that do not necessarily involve IL-1
production. In fact, caspase-1 is able to directly process pro-caspase-3 to its active form (Tewari et al., 1995), and it is also involved in the processing of IL-18 (Akita et al., 1997). Moreover, there is evidence that caspase-1 is an apical caspase in neuronal cell death pathways, being involved in Bid cleavage and caspase-3 activation following an ischemic insult (Benchoua et al., 2001; Zhang et al., 2003b). In conclusion, our data suggest that early production of IL-1
following ischemic stroke is not associated with caspase-1 activation. By contrast, MMPs appear to contribute to cytokine processing thus representing a crucial upstream signal for the induction of neuroinflammatory responses. This strengthens the involvement of MMPs in the development of tissue damage during the early phases after stroke and emphasizes their potential as useful pharmacological targets for the treatment of ischemia-induced neurodegeneration. Acknowledgments Partial finantial support from Italian Ministry of University and Research (PRIN prot. 2004053099_004 to G.B.) is gratefully acknowledged. Mr. Guido Fico is gratefully acknowledged for skillful technical assistance. References Akita, K., Ohtsuki, T., Nukada, Y., Tanimoto, T., Namba, M., Okura, T., Takakura-Yamamoto, R., Torigoe, K., Gu, Y., Su, M. S., Fujii, M., Satoh-Itoh, M., et al. (1997). Involvement of caspase-1 and caspase-3 in the production and processing of mature human interleukin 18 in monocytic THP.1 cells. J. Biol. Chem. 272, 26595–26603. MMPS MODULATE IL-1
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