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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
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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: lipocortin 1 • receptor • neutrophil • FPR • fMLP
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). 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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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
).
|
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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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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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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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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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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 
and macrophage inflammatory protein 1 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
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.
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ACKNOWLEDGMENTS |
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Received for publication March 23, 2001.
Revision received July 2, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 C. Bandeira-Melo, A. G. C. Bonavita, B. L. Diaz, P. M. R. e Silva, V. F. Carvalho, P. J. Jose, R. J. Flower, M. Perretti, and M. A. Martins A Novel Effect for Annexin 1-Derived Peptide Ac2-26: Reduction of Allergic Inflammation in the Rat J. Pharmacol. Exp. Ther., June 1, 2005; 313(3): 1416 - 1422. [Abstract] [Full Text] [PDF]
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P. Maderna, S. Yona, M. Perretti, and C. Godson Modulation of Phagocytosis of Apoptotic Neutrophils by Supernatant from Dexamethasone-Treated Macrophages and Annexin-Derived Peptide Ac2-26 J. Immunol., March 15, 2005; 174(6): 3727 - 3733. [Abstract] [Full Text] [PDF]
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 C. D. Filippo, R. Marfella, S. Cuzzocrea, E. Piegari, P. Petronella, D. Giugliano, F. Rossi, and M. D'Amico Hyperglycemia in Streptozotocin-Induced Diabetic Rat Increases Infarct Size Associated With Low Levels of Myocardial HO-1 During Ischemia/Reperfusion Diabetes, March 1, 2005; 54(3): 803 - 810. [Abstract] [Full Text] [PDF]
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 R. C. O. Zanardo, M. Perretti, and J. L. Wallace Annexin-1 is an endogenous gastroprotective factor against indomethacin-induced damage Am J Physiol Gastrointest Liver Physiol, March 1, 2005; 288(3): G481 - G486. [Abstract] [Full Text] [PDF]
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 M. Perretti and R. J. Flower Annexin 1 and the biology of the neutrophil J. Leukoc. Biol., July 1, 2004; 76(1): 25 - 29. [Abstract] [Full Text] [PDF]
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S. Ernst, C. Lange, A. Wilbers, V. Goebeler, V. Gerke, and U. Rescher An Annexin 1 N-Terminal Peptide Activates Leukocytes by Triggering Different Members of the Formyl Peptide Receptor Family J. Immunol., June 15, 2004; 172(12): 7669 - 7676. [Abstract] [Full Text] [PDF]
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F. N. E. Gavins, S. Yona, A. M. Kamal, R. J. Flower, and M. Perretti Leukocyte antiadhesive actions of annexin 1: ALXR- and FPR-related anti-inflammatory mechanisms Blood, May 15, 2003; 101(10): 4140 - 4147. [Abstract] [Full Text] [PDF]
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 M. Perretti and F. N. E. Gavins Annexin 1: An Endogenous Anti-Inflammatory Protein Physiology, April 1, 2003; 18(2): 60 - 64. [Abstract] [Full Text] [PDF]
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M. Perretti, F. Ingegnoli, S. K. Wheller, M. C. Blades, E. Solito, and C. Pitzalis Annexin 1 Modulates Monocyte-Endothelial Cell Interaction In Vitro and Cell Migration In Vivo in the Human SCID Mouse Transplantation Model J. Immunol., August 15, 2002; 169(4): 2085 - 2092. [Abstract] [Full Text] [PDF]
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