Formyl-Peptide Receptor 2/3/Lipoxin A4 Receptor Regulates Neutrophil-Platelet Aggregation and Attenuates Cerebral Inflammation: Impact for Therapy in Cardiovascular Disease

Abstract

Background: Platelet activation at sites of vascular injury is essential for hemostasis, but it is also a major pathomechanism underlying ischemic injury. Because anti-inflammatory therapies limit thrombosis and antithrombotic therapies reduce vascular inflammation, we tested the therapeutic potential of 2 proresolving endogenous mediators, annexin A1 N-terminal derived peptide (AnxA1Ac2-26) and aspirin-triggered lipoxin A4 (15-epi-lipoxin A4), on the cerebral microcirculation after ischemia/reperfusion injury. Furthermore, we tested whether the lipoxin A4 receptor formyl-peptide receptor 2/3 (Fpr2/3; ortholog to human FPR2/lipoxin A4 receptor) evoked neuroprotective functions after cerebral ischemia/reperfusion injury.

Methods and results: Using intravital microscopy, we found that cerebral ischemia/reperfusion injury was accompanied by neutrophil and platelet activation and neutrophil-platelet aggregate formation within cerebral microvessels. Moreover, aspirin-triggered lipoxin A4 activation of neutrophil Fpr2/3 regulated neutrophil-platelet aggregate formation in the brain and inhibited the reactivity of the cerebral microvasculature. The same results were obtained with AnxA1Ac2-26 administration. Blocking Fpr2/lipoxin A4 receptor with the antagonist Boc2 reversed this effect, and treatments were ineffective in Fpr2/3 knockout mice, which displayed an exacerbated disease severity, evidenced by increased infarct area, blood-brain barrier dysfunction, increased neurological score, and elevated levels of cytokines. Furthermore, aspirin treatment significantly reduced cerebral leukocyte recruitment and increased endogenous levels of aspirin-triggered lipoxin A4, effects again mediated by Fpr2/3.

Conclusion: Fpr2/lipoxin A4 receptor is a therapeutic target for initiating endogenous proresolving, anti-inflammatory pathways after cerebral ischemia/reperfusion injury.

Keywords: inflammation; ischemia reperfusion injury; stroke, ischemic.

Figure 1

Cellular responses are exacerbated in the cerebral microcirculation of Fpr2/3^−/− mice following I/R. Wild type (WT, C57BL/6) and Fpr2/3^−/− mice were subjected to MCAo for 60 min, followed by 4h or 24 h reperfusion. Leukocyte and platelet recruitment in the cerebral microcirculation was quantified in terms of: A) number (no.) of adherent leukocytes (cells stationary ≥ 30 sec) and B) number of adherent platelets (cells stationary for ≥ 2 sec). Representative intravital fluorescence microscopy video stills at 24 h showing few cellular interactions in the cerebral microcirculation of C) WT sham mice and D) Fpr2/3^−/− sham mice, or increased leukocytes and platelets in E) WT mice and further heightened increase in F) Fpr2/3^−/− mice. Images taken on an Olympus BW61WI microscope, magnification × 40. Data are mean ± SEM of 8 mice per group. *p < 0.05 & ***p < 0.001 vs. control. ^#p < 0.05 & ^###p < 0.001 vs. genotype at same time point.

Figure 2

Lack of Fpr2/3 leads to increased tissue damage in the brain following I/R. Wild type (WT, C57BL/6) and Fpr2/3^−/− mice were subjected to MCAo for 60 min, followed by 24 h reperfusion. A) Neurological score was assessed and brains removed to quantify B) infarct volume. In a separate cohort of animals, brain homogenate samples were taken. C) Blood brain barrier dysfunction was measured in right and left hemispheres from mice using Evan’s blue (EB) dye extraction. D) Myeloperoxidase (MPO) was measured in whole brain homogenates. **p < 0.01 & ***p < 0.001 vs. control. ^###p < 0.001 vs. genotype at same time point.

Figure 3

Inflammatory responses are augmented in Fpr2/3^−/− mice. Wild type (WT, C57Bl/6) and Fpr2/3^−/− mice were subjected to MCAo for 60 min, followed by a 24 h reperfusion, after which plasma and brain homogenate samples were taken. Cytokines were measured in plasma (A–E) and in homogenates from the left (L: ipsilateral) and right (R: contralateral) sides of the brain (F–J). Data are mean ± SEM of 8 mice per group. **p < 0.01 & ***p < 0.001 vs. same genotype right side. ^###p < 0.001 vs. WT left side.

Figure 4

Neutrophil Fpr2/3^−/− is key for NPAs in I/R injury. Wild type (WT, C57Bl/6) and Fpr2/3^−/− mice were subjected to MCAo for 60 min, followed by 24 h reperfusion. A) Plasma TXB₂ levels were measured. B) representative image of NPAs in cerebral I/R as seen with confocal intravital microscopy (Neutrophil: red; platelets: green). C) neutrophil/platelet aggregates (NPAs) were quantified in sham and I/R WT and Fpr2/3^−/− mice. D) Adoptive transfer experiments were performed by injecting CFSE-labeled platelets from donor mice (P) into recipient mice (i.e. KO-P = platelets isolated from Fpr2/3^−/− donor mice), and assessed via intravital microscopy. Data are mean ± SEM of 6–8 mice per group. ***p < 0.001 vs. sham. ^###p < 0.001 vs. control

Figure 5

Protective effects of ASA are inhibited in Fpr2/3^−/− mice. Wild type (WT, C57Bl/6) and Fpr2/3^−/− mice were subjected to MCAo for 60 min, followed by 24 h reperfusion. Vehicle (V. saline) or ASA (150 mg/kg) were administered 60 min prior to I/R. A+E) Plasma ATL levels; B+F) ATL levels in whole brain homogenates; C+G) adherent leukocytes and D+H) NPAs were measured in WT and Fpr2/3^−/− mice. Data are mean ± SEM of 6–8 mice per group. **p < 0.01 & ***p < 0.001 vs. vehicle. ^###p < 0.001 vs. ASA.

Figure 6

Schematic overview of the important anti-inflammatory role that Fpr2/3 plays in abrogating I/R-induced cerebrovascular injury. I/R leads to the production of Fpr2/-mediated formation of neutrophil/platelet aggregates (NPAs), with neutrophil Fpr2/3 mediating the signal. Aggregates allow the biosynthesis of LXA4 via neutrophil 5-lipoxygenase (5-LOX) and platelet 12/15-LOX. High levels of aspirin-triggered lipoxin (ATL) are also generated during I/R. The effect of ATL on the cerebral microcirculation was absent in mice that lack the Fpr2/3, but pharmacological targeting of the Fpr2/3 (with AnxA1_Ac2–26 or ATL) can resolve these inflammatory effects. The administration of aspirin (ASA) is able to produce ATL via COX2 acetylation, which in turn acts on Fpr2/3 and monitors the vascular permeability in the brain. In summary, Fpr2/3 (and we propose the human counterpart ALX/FPR2) promotes the biosynthesis and production of lipoxins and ATL, which in turn regulates the inflammatory process and restores homeostasis in the brain.