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(The FASEB Journal. 2001;15:2247-2256.)
© 2001 FASEB

Annexin 1 peptides protect against experimental myocardial ischemia-reperfusion: analysis of their mechanism of action

MYLINH LA1, MICHELE D’AMICO*, SILVIO BANDIERA, CLARA DI FILIPPO*, SONIA M. OLIANI{dagger}, FELICITY N. E. GAVINS, RODERICK J. FLOWER and MAURO PERRETTI

The William Harvey Research Institute, Charterhouse Square, London EC1M 6BQ, United Kingdom;
* Department of Experimental Medicine, Section of Pharmacology L. Donatelli, Second University of Naples, 80138 Naples, Italy; and
{dagger} Department of Biology, IBILCE-UNESP, Sao José do Rio Preto, SP, Brazil

1Correspondence: Department of Biochemical Pharmacology, The William Harvey Research Institute, Charterhouse Square, London EC1M 6BQ, United Kingdom. E-mail: m.la{at}mds.qmw.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Myocardial reperfusion injury is associated with the infiltration of blood-borne polymorphonuclear leukocytes. We have previous described the protection afforded by annexin 1 (ANXA1) in an experimental model of rat myocardial ischemia-reperfusion (IR) injury. We examined the 1) amino acid region of ANXA1 that retained the protective effect in a model of rat heart IR; 2) changes in endogenous ANXA1 in relation to the IR induced damage and after pharmacological modulation; and 3) potential involvement of the formyl peptide receptor (FPR) in the protective action displayed by ANXA1 peptides. Administration of peptide Ac2–26 at 0, 30, and 60 min postreperfusion produced a significant protection against IR injury, and this was associated with reduced myeloperoxidase activity and IL-1ß levels in the infarcted heart. Western blotting and electron microscopy analyses showed that IR heart had increased ANXA1 expression in the injured tissue, associated mainly with the infiltrated leukocytes. Finally, an antagonist to the FPR receptor selectively inhibited the protective action of peptide ANXA1 and its derived peptides against IR injury. Altogether, these data provide further insight into the protective effect of ANXA1 and its mimetics and a rationale for a clinical use for drugs developed from this line of research.—La, M., D’Amico, M., Bandiera, S., Di Filippo, C., Oliani, S. M., Gavins, F. N. E., Flower, R. J., Perretti, M. Annexin 1 peptides protect against experimental myocardial ischemia-reperfusion: analysis of their mechanism of action.


Key Words: lipocortin 1 • receptor • neutrophil • FPR • fMLP


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
REPERFUSION OF ISCHEMIC tissue can give rise to an inflammatory response that ultimately results in tissue injury. This phenomenon has been referred to as ‘reperfusion injury’ and is characterized by a complex series of events, including infiltration of polymorphonuclear leukocytes (PMN) (1 , 2) . The injuring role of PMNs in ischemia-reperfusion (IR) damage was first suggested by pioneering studies performed in animals depleted of circulating PMNs (3 , 4) and, more recently, by studies targeting the role of specific adhesion molecules involved in the leukocyte-vascular endothelium interaction (5 6 7) .

Activated PMNs, adherent to microvessels or infiltrated into the myocardium, can contribute to tissue damage by several mechanisms such as 1) release of free radicals after respiratory burst from the NADPH oxidase (O2-; OH-); 2) release of proteolytic enzymes (elastase, cathepsin G and proteinase), 3) stimulation of cytokines release from surrounding cells, thus exacerbating the process of leukocyte recruitment (8 , 9) . Finally, plugging of capillaries by PMN contributes to the no-flow phenomenon (10) . Therefore, inhibition of PMN accumulation to the myocardium or regulation of PMN activation is an obvious target for the development of novel therapies for myocardial ischemic injury observed after reperfusion.

Annexin 1 (ANXA1; previously referred to as lipocortin 1) is a member of the annexin superfamily of proteins that is endowed with a potent leukocyte antimigratory activity (11) . At least 13 distinct mammalian proteins have been described (12) . Structurally, annexins are characterized by having a core of four or eight conserved repeats, each containing ~70 amino acids. This core is attached to an amino-terminal segment that is unique for each member of the annexin family and is thought to be responsible for each annexin’s specific biological function(s) (13) . Administration of human recombinant ANXA1 reduces PMN extravasations in several animal models of acute inflammation, including a model of reperfusion injury in the rat small intestine (14 , 15) .

We recently described a beneficial effect of human recombinant ANXA1 in a model of rat myocardial IR injury (16) . In this experimental setting, ANXA1 reduced tissue necrosis and preserved the integrity of the myocardium after IR injury. The protective effect of ANXA1 was dose dependent and mirrored by a reduction in PMN extravasations (16) . The mechanistic and molecular basis for ANXA1 action has remained elusive, but the existence of specific annexin binding sites on human and rat neutrophils and monocytes has been reported (17 , 18) . A recent in vitro study has shown that ANXA1-derived peptide activates the formyl peptide receptor (FPR) on neutrophils (19) . FPR is a member of the seven trans-membrane domain, G-protein-linked receptor superfamily (20) ; its activation by bacterial products like N-formyl-Met-Leu-Phe (fMLP) initiates responses such as chemotaxis, superoxide production, and cell degranulation (20) .

Here we assessed the potential protective effect of peptides drawn from the ANXA1 NH2 terminus (peptides Ac2–26, Ac2–12, and Ac2–6) in a model of rat myocardial IR. Second, we have investigated the mechanism underlying myocardial protection, including the involvement of the FPR, using a selective antagonist. Finally, the expression of endogenous ANXA1 in relation to the IR induced damage and after pharmacological modulation was examined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental model
The procedure followed was described in detail in our previous study (16) . The trachea of anesthetized rats was cannulated to maintain ventilation and rats were kept at a body temperature of 37–38°C by a homeothermic blanket. The right carotid artery was cannulated, and mean arterial blood pressure (MABP) and heart rate (HR) were continuously monitored on a Gould polygraph. The pressure rate index (PRI), a relative indicator of myocardial oxygen consumption, was calculated as the product of MAP and HR and expressed in mmHg/min/103 (21) . The right jugular vein was cannulated for the administration of drugs. A left thoracotomy was performed and the pericardium was removed to expose the heart. A fine silk ligature was placed around the left anterior descending coronary artery (LADCA); after 30 min of equilibration, the LADCA was occluded for 25 min, followed by 2 h of reperfusion.

Measurement of area at risk (AR) and of infarct size (IS)
Two hours after the reperfusion period, the LADCA was reoccluded, and the following parameters were determined using sequential staining procedure as described previously (16) : AR was determined using Evans blue dye (1 ml of 2% wv-1); infarcted tissue was determined using p-nitro-blue tetrazolium (0.5 mg ml-1, 20 min at 37°C). The IS or necrotic tissue was calculated as a function of the AR mass (IS/AR) and total left ventricular weight (IS/lV) as described previously (16 , 22) .

Experimental groups
ANXA1 peptides were administered at doses previously validated in rodent models of leukocyte interaction with injured microvessels (15 , 23) , then 0.5 and 1 mg/kg (165 nmol/kg and 330 nmol/kg) for peptide Ac2–26, 1 mg/kg for peptides Ac2–12, scrambled Ac2–12 and Ac2–6 (700 nmol/kg, 700 nmol/kg, and 1.7 µmol/kg, respectively). The FPR antagonist N-t-butoxycarbonyl-Phe-Leu-Phe-Leu-Phe (Boc2) was injected at 0.4 mg/kg (500 nmol/kg); the FPR agonist fMLP was given at 0.2 mg/kg. Human recombinant ANXA1 was administered at 5 µg per rat (25 µg/kg corresponding to 0.7 nmol/kg), previously shown to inhibit myocardial injury produced with the experimental protocol detailed above (16) . For comparative purposes, the effect of the nonselective melanocortin receptor agonist HP228 (24) was tested at 0.5 mg/kg dose. The PARP inhibitor 3-aminobenzamide [3-AB; an inhibitor of poly(ADP-ribose) synthase (25) ] was also assessed at a dose of 10 mg/kg, previously shown to be cardioprotective (26) . A group of sham-operated rats (sham) and of vehicle-treated rats (saline) were always used as a negative control. Unless otherwise stated, all drugs were administered intravenously (i.v.) at the beginning of the reperfusion phase (time 0 reperfusion). In selected experiments, peptide Ac2–26 (1 mg/kg i.v.) was given 30 or 60 min after reperfusion. Overall mortality was <7% throughout the entire study.

Measurement of tissue myeloperoxidase activity
Myocardial myeloperoxidase (MPO) activity was measured according to the method previously described (16) . The AR was homogenized in Tris buffer containing proteinase inhibitors (see below) and homogenates were centrifuged for 30 min at 4000 g at 4°C. An aliquot (20 µl) of the supernatant was then allowed to react with a solution of tetra-methyl-benzidine (1.6 mM) and 0.1 mM H2O2. The rate of change in absorbance was measured with a spectrophotometer at 620 nm.

Assessment of rat interleukin 1ß (IL-1ß) levels in injured myocardium
Rat IL-1ß level was quantified in the AR at the end of the reperfusion period using a specific ELISA that shows negligible (<1%) cross-reactivity with other rat cytokines and has high sensitivity (~detection limit 9 pg/ml) (data furnished by the manufacturer). Tissue homogenates (50 µl) were assayed and compared with a standard curve constructed with 0–500 pg/ml rat IL-1ß.

Fixation, processing, and embedding for transmission electron microscopy
Preparation of the tissue sections for electron microscopy analysis was similar to a recently published protocol (16) . Fragments of the left ventricle of the heart were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde, 0.1 M sodium cacodylate buffer (pH 7.4) for 24 h at 4°C, then washed in sodium cacodylate, dehydrated through a graded series of ethanol, and embedded in LR Gold (London Resin Co., Reading, Berkshire, UK). Sections (~90 nm thick) were cut on an ultramicrotome (Reichert Ultracut; Leica, Austria) and placed on nickel grids for immunogold labeling.

Postembedding immunogold labeling
To detect ANXA1, we used an established immunogold staining procedure (27) . Fragments of the heart were stained with uranyl acetate (2% w/v in distilled water), dehydrated through increasing concentrations of ethanol (70–100%), and embedded in LR Gold resin. Ultrathin sections were prepared and incubated with the following reagents at room temperature: 1) 0.1 mol/l phosphate buffer containing 0.1% egg albumin (PBEA); 2) 2.5% normal rabbit serum in PBEA for 1 h; 3) a sheep polyclonal antibody termed LCPS1, raised against the amino-terminal peptide of human ANXA1 (peptide Ac2–26) (27) and used at a final dilution of 1:300 in PBEA. 4) Normal rabbit serum was used as control (1:300 final dilution); 5) after five washes (3 min each) in PBEA, a donkey anti-sheep IgG (Fc fragment specific) Ab (1:50 in PBEA) conjugated to 15 nm colloidal gold (British Biocell, Cardiff, UK) was added; 6) after 1 h at 4°C, sections were washed extensively in PBEA, then in distilled water. Ultrathin sections were stained with uranyl acetate and lead citrate before examination on a Zeiss electron microscope (Hertfordshire, UK).

Western blotting
In separate experiments, sham or infarcted hearts were infused with saline to remove excess blood and the left ventricles were homogenized in Tris-HCl 50 mM (pH 7.2) containing leupeptin 1 µM, pepstatin A 1 µM, PMSF 200 µM; total protein concentration was determined according to Bradford (28) . To detect ANXA1, protein extract (25 µg) from sham as well as animals subjected to IR and treated with either saline or Ac2–26 were loaded per lane onto a 12% SDS-PAGE for electrophoresis together with appropriate molecular weight markers and transferred to ECL Hybond nitrocellulose membrane. Reversible protein staining of the membranes with 0.1% Ponceau S in 5% acetic acid was used to verify even protein transfer. Membranes were incubated overnight in 5% nonfat dry milk together with a monoclonal antibody raised against full-length human ANXA1 [1:5000; reactive with the rat species, mAb 1B (29) ] in phosphate-buffered saline with 0.1% Tween 20 (PBST). This was followed by 30 min washing with PBST and incubation for 60 min at room temperature with peroxidase-conjugated goat anti-mouse IgG (1:2000). Membranes were again washed twice for 15 min with PBST and immunoreactive proteins were detected using an ECL kit from Amersham. Relative band intensity was quantified using NIH image software 1.62.

Materials
Peptides Ac2–26 (acetyl-AMVSEFLKQAWIENEEQEYVVQTVK, Mr 3,050), Ac2–12 (acetyl-AMVSEFLKQAW, Mr 1,424), scrambled Ac2–12 (acetyl-SVEQKMWALFA, Mr 1,424), and Ac2–6 (acetyl-AMVSE, Mr 600) were prepared by the Advance Biotechnology Center (The Charing Cross and Westminster Medical School, London) by using solid-phase stepwise synthesis. Purity was more than 90% as assessed by HPLC and capillary electrophoresis (data supplied by manufacturer). Human recombinant ANXA1 was provided by Dr. Egle Solito (Imperial College School of Medicine, London). Rat IL-1ß QuantikineTM ELISA was from R&D Systems (Abingdon, UK). HP228 was from Bachem (St. Helens, Merseyside, UK); all other chemicals were from Sigma-Aldrich Ltd. (Poole, Dorset, UK).

Statistical analysis
All values are expressed as mean ± SE of mean, with (n) number of rats per group. Statistical analysis was assessed either by Student’s t test or one-way ANOVA where appropriate. A probability of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of ANXA1-derived peptides on myocardial IR
Occlusion of the LADCA and subsequent reperfusion produced a marked damage in the rat left ventricle that was reliably measured at the 2 h point (Fig. 1 ). Approximately 50% of the left ventricle remained unstained by Evans blue underlying the AR (Fig. 1A ); 50% of this portion of the ventricle was infarcted (Fig. 1B ). Sham operation of the rats produced a small degree of injury as measured at the 2 h point. Administration of peptide Ac2–26 immediately after the opening of the LADCA produced a dose-dependent protection against the myocardial injury as measured 2 h later (Fig. 1B , C ). Similarly, administration of peptide Ac2–12 also protected against myocardial injury but the scrambled sequence of Ac2–12 was not protective. In contrast, the shorter peptide Ac2–6 produced no protection (Fig. 1B , C ). Occlusion of LADCA produced a significant decrease in MABP and PRI with no marked effects on HR in all groups studied (Table 1 ). Reperfusion of the LADCA reversed the decreased in MABP and PRI to almost basal levels in all groups. Administration of all three peptides did not alter these values (Table 1) .



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  		Figure 1. Effects of ANXA1-derived peptides on myocardial ischemia-reperfusion injury. Rats were treated i.v. with saline (1 ml/kg), peptide Ac2–26 (0.5 and 1 mg/kg), peptide Ac2–12 (1 mg/kg), scramble Ac2–12 (S, 1 mg/kg), or peptide Ac2–6 (1 mg/kg) at the end of the 25 min ischemic period. Tissues were analyzed 2 h after reperfusion; the area at risk (A), infarct size/area at risk (B), infarct size/left ventricle (C) were determined as described in Materials and Methods. A group of sham-operated animals (sham) was also evaluated. Data are means ± SE of n = 5–14 rats per group. #P < 0.05 vs. sham and *P < 0.05 vs. saline treatment.


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  		Table 1. Mean arterial blood pressure, heart rate, and pressure rate index during 25 min of coronary occlusion and 2 h reperfusion in rats subjected to various treatmentsa

To mimic the clinical situations where drugs are administered after myocardial infarction, we assessed the protective action of peptide Ac2–26 when administered 30 and 60 min after reperfusion. The greatest inhibition of IS/AR ratio produced by peptide Ac2–26 was measured after administration 30 min after reperfusion, whereas administration of the peptide at 60 min still conferred a significant degree of protection (Fig. 2 ).



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  		Figure 2. Time course of peptide Ac2–26 cardioprotective effect. Rats were given peptide Ac2–26 (1 mg/kg i.v.) immediately after the start of reperfusion (time 0), 30, and 60 min after reperfusion, and tissue was collected at 120 min to determine the infarct size/area at risk value as described in Materials and Methods. Infarct size was significantly reduced at all times vs. control (IS/AR value=55±2%, n=6). Data are mean ± SE of n = 4–6 rats per group. *P < 0.05 vs. control.

Insight into the mechanism of the protective action of ANXA1-derived peptides
We had previously shown that ANXA1 protects against myocardial IR injury by reducing PMN recruitment (16) . Therefore, we assessed the effect of peptide Ac2–26 on PMN recruitment to the myocardium by measuring MPO activity and the cytokine IL-1ß. Samples obtained from animals subjected to IR and treated only with saline had a markedly significant increase in MPO activity vs. sham (Fig. 3A ). MPO activity values were reversed to almost basal levels in animals treated with 1 mg/kg peptide Ac2–26 (Fig. 3A ). Figure 3B shows that ischemia-reperfusion injury induced a threefold augmentation (P<0.05) over the basal level of IL-1ß detected in the hearts of sham animals. Treatment with Ac2–26 also significantly reduced IL-1ß level in infarcted myocardium (Fig. 3B ).



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  		Figure 3. Myeloperoxidase (MPO) activity and IL-1ß levels in injured myocardial tissues. Rats were treated with saline (1 ml/kg) or peptide Ac2–26 (1 mg/kg) as in Fig. 1 legends; samples of the area at risk were collected at the end of the 2 h reperfusion period and assayed for MPO activity (A) and IL-1ß (B) levels. Tissues from sham-operated animals were also evaluated. Data are mean ± SE of n = 4–6 rats per group. #P < 0.05 vs. sham and *P < 0.05 vs. saline treatment.

The potential role of FPR in the protection produced by peptide Ac2–26 was then determined. Treatment with the FPR antagonist Boc2 significantly reversed the cardioprotective actions of peptide Ac2–26, peptide Ac2–12 and ANXA1, whereas injection of the FPR antagonist alone did not modify the degree of tissue damage (Fig. 4 , Fig. 5 ). Administration of these compounds did not alter the MABP, HR, or PRI (Table 2 ).



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  		Figure 4. Boc2 reversed the cardioprotective actions of ANXA1 and its NH2 terminus peptides. The effect of peptide Ac2–26 (0.5 mg/kg) and ANXA1 (25 µg/kg) administered alone or together with 0.4 mg/kg Boc2. Drugs were administered 25 min after ischemia and tissues were analyzed 2 h after reperfusion; the area at risk (A), infarct size/area at risk (B), and infarct size/left ventricle (C) were determined as described in Materials and Methods. Data are means ± SE of n = 5 rats per group. *P < 0.05 vs. saline treatment.



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  		Figure 5. Boc2 reversed the protection afforded by fMLP but had no effect on the actions of HP228 or 3-AB. The cardioprotective effect of HP228 (0.5 mg/kg), 3-AB (10 mg/kg), and N-formyl-Met-Leu-Phe (fMLP; 0.4 mg/kg) was investigated in the presence and absence of 0.2 mg/kg Boc2. Drugs were administered after 25 min ischemia; tissues were collected 2 h after reperfusion to determine the infarct size/area at risk as described in Materials and Methods. Data are mean ± SE of n = 4–14 rats per group. *P < 0.01 vs. saline treatment; #P < 0.01 vs. fMLP alone.


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  		Table 2. Mean arterial blood pressure, heart rate, and pressure rate index during 25 min of coronary occlusion and 2 h reperfusion in rats subjected to various treatmentsa

The selectivity of Boc2 inhibitory action was confirmed using drugs acting through different pathways. HP228, an agonist at several seven trans-membrane domain G-protein-linked melanocortin receptors (24) , produced a significant reduction in IR-induced damage; this effect was still present when coinjected with Boc2 (Fig. 5) . Similarly, the protection measured with the poly(ADP-ribose) synthase inhibitor 3-AB was unaffected by Boc2 (Fig. 5) . Administration of Boc2, HP228, 3-AB, and ANXA1 had no effect on MABP, HR, or PRI, as summarized in Table 3 .


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  		Table 3. Mean arterial blood pressure, heart rate, and pressure rate index during 25 min of coronary occlusion and 2 h reperfusion in rats subjected to various treatmentsa

Finally, activation of FPR by the agonist fMLP resulted in a significant protection against heart IR injury, and this was susceptible to Boc2 inhibition. Together, these data suggest that fMLP produced a protective effect similar to that displayed by peptide Ac2–26 and ANXA1 (Fig. 5) .

Expression of endogenous ANXA1
In the last part of this study we monitored endogenous ANXA1 expression. As expected (30) , little or no ANXA1 was detected in myocardial extracts prepared from naive or sham-operated rats (Fig. 6 A). However, animals subjected to IR and treated with saline expressed the characteristic ANXA1 doublet with the 34 kDa and 37-isoforms. This was apparently reduced in the hearts collected from rats treated with a cardioprotective dose of peptide Ac2–26 (Fig. 6A ). Figure 7 B represents the semi-quantitative densitometry analysis of the ANXA1 doublet, with a mean of >5 rats per group. Peptide Ac2–26 significantly reduced endogenous ANXA1 myocardial immunoreactivity, which was greatly augmented by the IR procedure.



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  		Figure 6. Expression of endogenous ANXA1 in myocardial samples. A) Western blot analysis of endogenous ANXA1 expression in sham (S), naive (N) animals, and those subjected to IR treated with either saline (C) or 1 mg/kg peptide Ac2–26 (P). B) Densitometric analysis for ANXA1 37 and 34 kDa isoforms. Values are mean ± SE of n = 5–8 rats per group. *P < 0.01 vs. sham treatment; #P < 0.01 vs. saline group.



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  		Figure 7. Presence of leukocytes in myocardial vessels after IR as determined by transmission electron microscopy. After the 2 h reperfusion period, leukocytes can be seen interacting with the myocardial vessel endothelial wall (arrow) and migrated into the perivascular tissue (arrowheads). Picture is representative of three distinct preparations. Erythrocytes (E); bar = 1 µm.

The localization of endogenous ANXA1 under these experimental conditions was determined by electron microscopy analysis. Indeed, visualization of the myocardial vessels confirmed the presence of leukocytes interacting with the endothelium and also in the subendothelial space already 2 h postreperfusion (Fig. 7) after the ischemic and reperfusion injury. The localization of endogenous ANXA1 under these experimental conditions was determined by immunocytochemical analysis. Intravascular neutrophils and cardiomyocytes showed modest ANXA1 immunoreactivity throughout the cytosol and the nucleus in sham-operated rats (Fig. 8a , b ). In contrast, neutrophils adherent to or migrated through the postcapillary endothelium were also strongly positive for ANXA1, as detected 2 h after IR (Fig. 8d ). Cardiomyocytes of infarcted hearts were also positive for the protein (Fig. 8c ). No labeling was detected in sections incubated with control nonimmune sheep serum (Fig. 8e , f ).



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  		Figure 8. Immunocytochemistry for ANXA1 in cardiomyocytes and extravasated neutrophils. Electron micrograph of a heart tissue stained with a polyclonal sheep serum raised against the specific ANXA1 NH2 terminus. a, b) Cardiomyocyte and neutrophil of sham-operated rats, respectively, show some immunoreactivity throughout the cytosol (arrowheads) and nucleus (arrows). c, d) After 2 h reperfusion, a greater proportion of ANXA1 immunoreactivity is detected in cardiomyocyte (cytosol, arrowheads; nucleus, arrows) and extravasated neutrophil. e, f) Absence of gold labeling in sections incubated with control nonimmune sheep serum as seen in cardiomyocytes and neutrophil, respectively. Pictures are representative of three distinct preparations. Bars = 1 µm in all panels.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study was undertaken to investigate the potential application of the recently reported cardioprotective property of ANXA1 (16) and to provide a molecular mechanism of action. We demonstrate here that short peptides derived from the ANXA1 NH2 terminus mimicked the effect of the parent protein and provide pharmacological evidence that activation of FPR is the mechanism underlying this cardioprotective activity.

ANXA1 (360 amino acids) is a 37 kDa member of the annexin superfamily of proteins (12) . In human (31) and rodent (27) neutrophils, the protein is contained predominantly within gelatinase granules and is externalized onto the neutrophil cell membrane after cell adhesion to endothelial cells. Once on the cell surface, ANXA1 acts by a mechanism yet to be clarified in order to reduce leukocyte extravasation (32) . These effects are mimicked by exogenous administration of human recombinant ANXA1 in both acute and chronic models of inflammation and leukocyte extravasation (14 , 33) . In these systems, ANXA1 anti-inflammatory effects are retained by short peptides derived from the unique amino-terminal region (15 , 34) . Since we have recently reported that ANXA1 can inhibit myocardial damage in an experimental model of IR injury (16) , this study began by determining the effect of ANXA1-derived peptides. Using peptide fragments derived from the amino-terminal region, we have identified that the pharmacophore responsible for cardioprotection lies within amino acids 2–12, and that their correct alignment is crucial to retain biological activity. These observations are consistent with other studies from our laboratory in which peptides Ac2–26 and Ac2–12 inhibited leukocyte migration in mesenteric microvessels, as determined by intravital microscopy (23) and leukocyte recruitment into the mouse peritonitis (35) . It is noteworthy that peptide Ac2–26 was also effective against IR injury when administered up to 60 min after reperfusion. Such a finding would suggest that peptide Ac2–26 (and, by extension, novel ANXA1 mimetics) may have the potential to be applied in clinical situations where drugs can be administered only after the reperfusion phase has begun.

It is not surprising that on a molar basis, ANXA1 was more potent than its NH2 terminus-derived peptides, as already observed with respect to leukocyte migration (36) . Besides pharmacokinetic implications, another possible reason is that full-length ANXA1 contains two biological active sites, the NH2 terminus domain and region 246–254, that correspond to antiflammin 2. This nonapeptide possesses potent anti-inflammatory activity (37) ; its target seems to be the neutrophil (38) .

The accumulation of PMN into the injured myocardium is a major cause of reperfusion injury (8 , 9) . PMN are recruited to the reperfused myocardium by chemotactic factors released by the myocardium during the ischemic period (9) . We have previously demonstrated that chemotactic factors such as tumor necrosis factor {alpha} and macrophage inflammatory protein 1{alpha} were significantly reduced in IR-injured hearts treated with ANXA1 (16) . In this study we monitored tissue levels of IL-1ß, another pleiotropic proinflammatory cytokine, finding that its expression was significantly increased by IR injury and reduced by administration of Ac2–26. These observations were coupled with the assessment of the presence of PMN in the myocardium, as achieved by measuring MPO activity (39) . We had demonstrated earlier that myocardial MPO activity increased before detectable tissue damage and that this augmentation was inhibited by ANXA1 administration (16) . Similar to our finding with the whole protein, treatment of rats with peptide Ac2–26 significantly reduced MPO activity in IR-injured hearts. These data, together with the ‘leukocyte detachment’ property demonstrated for peptide Ac2–12 and Ac2–26 (23) , point to the infiltrated/adherent leukocyte as the mechanism by which myocardial IR injury is reduced. Recently, ANXA1 was shown to inhibit changes in leukocyte integrin activation induced by different mediators (38) without affecting the expression of adhesion molecule on endothelial cells. Similar data have been obtained with peptide Ac2–26, previously shown to inhibit neutrophil-endothelial cell interaction when elicited with neutrophil but not endothelial cell activators (40) . Changes in leukocyte adhesion molecule expression can clearly affect this cell-to-cell interaction. Besides the studies mentioned above, ANXA1 has also been shown to affect L-selectin (CD62L) shedding (41) and to interfere with the binding of vascular cell adhesion molecule 1 to {alpha}4ß1 integrin in the U937 cell line (42) . The latter class of adhesion molecules governs neutrophil interaction with the cardiomyocyte (43) .

Despite its well-known antimigratory activity, the molecular mechanism for the action of ANXA1 has so far remained elusive. The existence of specific and saturable binding sites on human and rodent neutrophils and monocytes has been reported (17 , 44 , 45) , but their exact nature has yet to be identified. Walther et al. (19) used a series of in vitro assays to report the existence of a functional interaction between ANXA1-derived amino-terminal peptides and the formyl peptide receptor (acronym, FPR). Activation of FPR by fMLP leads to in vitro activation of neutrophils and monocytes/macrophages, an effect blocked by the competitive FPR antagonist Boc2 (20) . More recently, we have found a partial involvement of FPR in the antimigratory actions of ANXA1 and derived peptides (35) . Therefore, an important part of the present study was to determine whether endogenous FPR was functionally linked to the protective actions displayed by ANXA1 (16) and its amino terminal-derived peptides. The data obtained with the antagonist are quite conclusive and demonstrate, for the first time, an in vivo involvement of FPR in the cardioprotective properties of these compounds. Boc2 abrogated the protective effect displayed by i.v. injection of fMLP but had had no effect against either HP228 or 3-AB. The melanocortin agonist HP228 (46) was chosen because it activates a different class of seven trans-membrane domain G-protein-linked receptors, the melanocortin receptors. In our experimental setting, HP228 was cardioprotective in a Boc2-insensitive manner. Similarly, Boc2 did not affect the inhibitory properties of 3-AB. This drug was chosen because it bypasses the receptor stage; it inhibits poly(ADP-ribose) synthase and is known to be protective in this model of myocardial IR injury (26) .

Overall, these data provide in vivo relevance to the finding by Walther et al., (19) in the context of cardioprotection. However, in the in vitro study, FPR-activating activity was retained by peptides containing the region 19–25 (19) whereas, in our hands, the region spanning amino acids 2–12 retained biological activity and was sensitive to Boc2 treatment. Future studies are needed to address this apparent discrepancy in the sequence of the ANXA1 NH2 terminus required for FPR activation, though it is important to note that region 2–12 has recently been shown to be important for ANXA1 binding to endothelial cells and, hence, to localize the protein in the correct microenvironment to exert its inhibitory effect on leukocyte transmigration (47) . It will be interesting to see whether an FPR-like receptor expressed by the endothelium can also be involved in this complex scenario (48) .

Finally, we examined the expression of endogenous ANXA1 in this model. As expected (30) , the hearts of sham or naive animals did not express ANXA1. However, in analogy to other inflammatory conditions (27 , 49) , tissue infiltration by neutrophils brings about ANXA1 expression. In injured hearts, the protein was detected in the myocytes and more markedly in the neutrophils, as assessed by electron microscopy. This could be the result of de novo ANXA1 synthesis in infiltrated neutrophils as already demonstrated during experimental inflammation (27) . It is obscure whether the myocyte activates ANXA1 synthesis as well or whether the protein is somehow passed to it through a juxtacrine mechanism. This will be determined in future studies; nevertheless, these data show for the first time that myocardial injury brings about ANXA1 immunoreactivity. Endogenous ANXA1 appeared as a doublet (37 and 34 kDa bands), and this seemed to be modulated by treatment with peptide Ac2–26. These observations are all consistent with a recent study from our laboratory studying ANXA1 isoform expression in emigrating leukocytes during an acute inflammatory reaction (27) . The catabolism of ANXA1 is of physiological significance, as the 34 kDa fragment lacks anti-inflammatory activity (50) . The enzyme responsible for the catabolism of ANXA1 is currently unclear, but Ac2–26 might compete with the intact ANXA1 peptide at the enzyme level and thereby reduce ANXA1 catabolism. This may be at least contributory to the cardioprotective action displayed by peptide Ac2–26.

In conclusion, this investigation has pinpointed the active region within the amino-terminal domain of ANXA1 in an experimental model of myocardial infarct so that future drug development can be modeled on this region. In addition, we showed for the first time that peptide Ac2–26 was effective when administered up to 60 min after reperfusion, indicating the potential for application in clinical situations. Finally, we have proposed that ANXA1 and ANXA1-derived peptide interaction with a Boc2-sensitive receptor(s) is instrumental to the cardioprotective properties of these molecules.


   ACKNOWLEDGMENTS
 
This work was supported by the Joint Research Board of the Special Trustee of St. Bartholomew’s Hospital (grant XMKZ) and the British Heart Foundation (Ph.D. studentship FS/2000076 to F.N.E.G.). M.P. is a postdoctoral fellow of the Arthritis Research Campaign; R.J.F. is a Principal Research Fellow of the Wellcome Trust, UK. S.M.O was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil. The authors thank Dr. Kai Zacharowski for his expert advice.

Received for publication March 23, 2001. Revision received July 2, 2001.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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