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Annexin 1 system protects against murine cerebral ischaemia-reperfusion injury Stroke is the second leading cause of mortality worldwide with an estimated 9.5% of all deaths. Thus, there is a clear a need of new effective treatments. Since there is evidence for inflamm atory mechanisms being operative in stroke, we tested the potential protective actions of the anti-inflammatory annexin 1 (AnxA1) system. A model of focal cerebral ischaemia-reperfusion (IR) associated with intravital microscopy to monitor leukocyte-endothelial cell interactions in cerebral cortical vessels was applied. Male littermate wild type (WT) and AnxA1 null mice (25-30g) were used. Middle cerebral artery occlusion (MCAO) was induced for 1 h followed by 24 h reperfusion. At the start of reperfusion, drug/vehicle treatment occurred. The annexin 1 mimetic peptide Ac2-26 (Ac-AMVSEFLKQAWFIENEEQEYVQTVK: 100 μg, equivalent to 33 nmol), whole protein annexin 1 (1 μg, equivalent to 28 pmol), the formyl peptide receptor (FPR) antagonist Boc2 (N-tbutyloxycarbonyl-Phe-DLeu-Phe-DLeu-Phe: 10 μg, equivalent to 12 nmol) were administered. At the end of the 24 h reperfusion period, neurological deficit, infarct volume and leukocyte-endothelial cell interactions (LEI), e.g. leukocyte rolling and adhesion were determined. All values are expressed as mean SE of mean, with number (n) of animals per group where stated. Statistical tests performed were either by Student t test (2 groups) or by 1-way analysis of variance (ANOVA) followed by Bonferroni post hoc test (more than 2 groups). In all cases, a probability value of P less than 0.05 was considered significant. In contrast to sham mice, IR significantly increased LEI in both WT and AnxA1 null mice. The number of adherent leukocytes was increased compared to sham counterparts in both WT (472±77 vs. 0 cells, P<0.05, n=6 mice) and AnxA1 null mice (728±113 vs. 33±33 cells, P<0.05, n=6 mice). Administration of human recombinant AnxA1 (1 m g, 3 times) to null mice restored the WT phenotype. The pharmacological exploitation o f the AnxA1 system was assessed by testing the effect of N-terminal peptide Ac2-26. Given at time 0, 6 and 18 h peptide Ac2-26 (100 m g) inhibited infarct volume (33±2% vs. 45±6% in vehicle-treated animals; P<0.05, n=6 mice). This protective effect was mirrored by reduced leukocyte adhesion, compared to vehicle-treated animals (259±40 vs. 472±77 cells, P<0.05, n=6 mice). The FPR antagonist Boc2, inactive when given to IR-treated WT or annexin 1 null mice, prevented the inhibitory effect of peptide Ac2-26. These findings demonstrate that a bioactive peptide spanning the pharmacophore of the anti-inflammatory mediator AnxA1 caused significant cerebral protection by activating a receptor of the FPR family. Endogenous annexin 1 exerts a tonic inhibitory role on these cerebral vessels as determined with the AnxA1 null mouse. These 3 results shed light on the molecular mechanism underlying the tissue-protective effects of AnxA1 and on its potential for therapeutic development in cerebral ischaemia.
This work was supported by the British Heart Foundation UK Junior Fellowship. |
