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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 26, 2004 as doi:10.1096/fj.04-2178fje. |
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* Centre of Biochemical Pharmacology, The William Harvey Research Institute, Charterhouse Square, London, UK;
Department of Experimental Medicine, Second University of Naples, Naples, Italy; and
Department of Biology, IBILCE–UNESP, Sao José do Rio Preto, São Paulo, Brazil
1 Correspondence: The William Harvey Research Institute, Bart’s and the London, Queen Mary School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK. Email: m.perretti{at}qmul.ac.uk
SPECIFIC AIMS
There is renewed interest in endogenous inhibitory/counter-regulatory pathways that operate during the host inflammatory response to guarantee its time-dependency and thereby promote the phase of resolution. One of these mediators is the leukocyte-derived 37kDa protein annexin 1 (ANX-A1). When administered to experimental animals, ANX-A1 and its peptido-mimetics (including the N-terminal derived peptide Ac2-26) affect several facets of the innate inflammatory response, spanning from inhibition of neutrophil and plasma protein extravasation, to inhibition of macrophage phagocytosis and promotion of neutrophil apoptosis. ANX-A1 and its bioactive peptides also exert protection in rat models of acute myocardial ischemia-reperfusion (I/R) injury.
Recent interest about the ANX-A1 field has come from the notion that specific G-protein-coupled receptors, members of the formyl-peptide receptor (FPR) family, appear to mediate its anti-inflammatory actions. In the present study we have used an acute myocardial infarct model in the mouse to assess: 1) the protective actions of the bioactive ANX-A1 peptide (termed peptide Ac2-26) in wild-type (WT) mice; 2) its effects in FPR knockout (KO) mice; and 3) information about the mechanisms associated with peptide Ac2-26-mediated cardioprotection.
PRINCIPAL FINDINGS
1. Peptide Ac2-26 protects against acute myocardial I/R in WT and FPR KO
Occlusion of the left anterior descending coronary artery for 25 min, followed by reopening led to acute myocardial injury detected as early as 60 min post-reperfusion: 51.2 ± 4.3% of the area at risk (AAR) was infarcted vs. values of 5 ± 1% in the sham group. Administration of peptide Ac2-26 at a dose of 1 mg/kg (corresponding approximately to 30 µg or 9 nmol per mouse) immediately after reperfusion afforded significant cardioprotection (
50% reduction of the infarct, n=6, P<0.05). Gene deficiency in mouse FPR did not affect cardioprotection afforded by peptide Ac2-26. Administered at 1 mg/kg, peptide Ac2-26 markedly attenuated tissue damage by 45% and was not dissimilar from the protection afforded in WT mice. Boc2 compound, a putative FPR antagonist, abrogated the inhibitory effect of peptide Ac2-26 both in WT and FPR KO mice.
2. Peptide Ac2-26-mediated cardioprotection and markers of tissue inflammation
Myocardial injury was associated with an intense accumulation of bloodborne polymorphonuclear cells (PMN) as detected by measuring myeloperoxidase activity into the area at risk. Figure 1
A shows this increase in equivalent PMN numbers post-I/R in respect to the minimal damage measured in sham operated animals. Administration of the protective dose of peptide Ac2-26 (1 mg/kg) significantly attenuated these values as measured 60 min post-reperfusion (Fig. 1A
). The degree of inhibition was similar in WT and FPR KO mice, with calculated net inhibitions of 55% and 45%, respectively (n=6, not significant). Histological analyses demonstrated I/R-dependent presence of leukocytes both on the endothelium of myocardial blood vessel as well as into the perivascular connective tissue (Fig. 1B
). In line with the myeloperoxidase assay, peptide Ac2-26 qualitatively reduced leukocyte recruitment. Figure 1C
shows a representative vessel in which some intravascular, but few extravasated, cells were seen. Similar results were obtained in FPR KO mice. Changes in the extent of leukocyte interaction with myocardial vessels were associated with parallel changes in the tissue CXC chemokine KC (CXCL1) contents. Treatment with peptide Ac2-26 nearly abolished I/R-induced increase in KC levels both in WT and FPR KO mice (Fig. 1D
).
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3. Mouse ALX and circulating PMN
Expression of the lipoxin A4 receptor (or ALX), related to FPR, in the heart of WT and FPR KO mice was monitored. Genomic DNA analyses confirmed the expression of the correct genes in both mouse genotypes (Fig. 2
A). mRNA for both receptors was also detected in naive myocardial samples. Figure 2A
, right panel, shows a representative RT-PCR from a WT mouse and an apparent increase in ALX message after I/R. However, analysis of few samples showed that this was not a consistent result (Fig. 2B
). When the AXL agonist W peptide (Trp-Lys-Tyr-Met-Val-met) was injected at doses of 5 and 10 µg per mouse (corresponding to 5.5 and 11 nmol per mouse, or 0.15-0.3 mg/kg) a dose-dependent cardio-protective effect was evident with similar degree of protection in WT and FPR KO mice (Fig. 2C)
. A stable lipoxin A4 agonist, termed ATLa-ME (administered at the dose of 5 µg per mouse, equivalent to 9 nmol) produced 60% cardioprotective effect.
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Treatment of mice with an antineutrophil serum (reducing circulating neutrophils by >95%) did not change the acute myocardial damage as measured 60 min post-reperfusion. The peptide Ac2-26 cardioprotective effect was no longer detected in these neutropenic mice.
CONCLUSIONS AND SIGNIFICANCE
Two previous studies of myocardial I/R injury in the rat have described the ability of ANX-A1 and its peptidemimetics to afford cardioprotection, however the search for the potential receptor target mediating these effects has been elusive. Efficacy of the Boc2 antagonist, also used in the present study, led us to propose an involvement of a receptor of the FPR family. We have confirmed these results in the mouse and demonstrated a redundant role for FPR, since cardioprotection was maintained in FPR KO mice. In line with a recent study on I/R-induced leukocyte recruitment in the mouse mesentery, we provide evidence for an involvement of the FPR-related receptor ALX. This conclusion is based on the expression patterns and on the qualitative similarities between peptide Ac2-26 and two ALX agonists, W peptide and ATLa-ME.
Analysis of specific markers of tissue inflammation and injury indicated a potent inhibitory action for peptide Ac2-26, and these effects all appeared to be FPR independent. Comparative analysis of ANX-A1 physiopharmacological effects studied in several laboratories using distinct techniques and models, prompt us to suggest that activation of distinct receptors bring about the actions of this anti-inflammatory mediator in a tissue-specific fashion. Using a peritonitis model, we highlighted a fundamental role for mouse FPR in mediating the antimigratory properties of peptide Ac2-26, but this was only partially true for full length ANX-A1. However, direct observation of the leukocyte-endothelium interaction by intravital microscopy analysis of postischemic mesenteric venules demonstrated that a significant proportion of the postischemic effect of peptide Ac2-26 was unaltered in FPR KO mice. Finally, we report that FPR is not involved in the mouse acute myocardial injury model employed in this study. The same may be applicable to the effects of these peptides on the isolated pituitary. Thus, in analogy to other mediators, it appears that different ANX-A1 receptors mediate specific inhibitory actions in different tissue sites and organs (Fig. 3
). The novel data here reported may help to exploit the ANX-A1 counterregulatory biochemical system for the development of mimetics endowed with protective actions on acute heart injury.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2178fje;
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