Selective FPR2 Agonism Promotes a Proresolution Macrophage Phenotype and Improves Cardiac Structure-Function Post Myocardial Infarction

Figures

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  Figure 1. Signaling Profile of BMS-986235 in HEK293 Cells Expressing Human FPR2 and FPR1 The ability of BMS-986235 to engage different signaling pathways was assessed using bioluminescence resonance energy transfer (BRET) biosensors detecting the activation of (A) Gαi1 (B) Gαi2, (C) Gαi3, (D) GαoA, and (E) GαoB; the interaction of effectors with (F) Gα12 and (G) Gα13; and the recruitment of (H) β-arrestin1 and (I) and β-arrestin2 to the plasma membrane. HEK293 cells expressing human formyl peptide receptor 2 (FPR2) or FPR1 were stimulated with BMS-986235, and modulation of the BRET signals from the different biosensors was recorded. Data represent the mean ± SEM of 3 independent experiments. ND = not determined.

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  Figure 2. Regulation of Inflammatory Cell Function by BMS-986235 (A) Enhancement of zymosan phagocytosis by BMS-986235 was assessed in BioGel-elicited peritoneal macrophages from wild-type, FPR1 knockout (KO), and FPR2 KO mice. (B) Stimulation of oxidative burst activity was evaluated in neutrophil-like HL-60, HL-60 FPR1 KO, and HL-60 FPR2 KO cell lines. (C) Chemotactic responses to BMS-986235 were evaluated in differentiated HL-60, HL-60 FPR1 KO, and HL-60 FPR2 KO cell lines. (D) Inhibition of chemotaxis toward serum amyloid A by BMS-986235. Curves were fit using least squares method of nonlinear regression (GraphPad Prism).The half maximal effective concentration (EC50) value for the HL-60 FPR1 KO data shown in C assumed a bell-shaped curve and was calculated from the stimulatory phase of the response. Dotted lines indicate no curve fit. Data represent the mean ± SEM of 3 independent experiments.

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  Figure 3. In Vitro mRNA Expression of Cytokines and FPR2 in Isolated Human Blood (A) Interleukin (IL)-10, (B) monocyte chemoattractant protein (MCP)-1, (C) IL-6, and (D) formyl peptide receptor 2 (FPR2). Whole blood from 20 consenting donors was processed as described in the Supplemental Material and Methods. Each data point represents the mean of at least triplicate measurements per donor. The mean value is depicted by the solid line relative to the distribution of data points for each concentration. Dunnett’s vs vehicle: ∗P < 0.05, ∗∗∗P < 0.001. mRNA = messenger RNA.

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  Figure 4. Apoptosis of Isolated Human Neutrophils Neutrophils were incubated with dimethyl sulfoxide (DMSO) (0.02%), serum amyloid A (SAA) (10 μg/mL), or the combination of BMS-986235 (2 μM, 15-minute pretreatment) and SAA (10 μg/mL). The relative ratios of viable, apoptotic, and dead cells were determined for each treatment condition using annexin V and propidium iodide staining followed by flow cytometry. The dashed lines depict the mean of viable, apoptotic, and dead neutrophils for DMSO reference control. Data represent the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, unpaired t test.

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  Figure 5. Acute IL-10 Response and Post-MI Infarct Phenotype in Mouse (A) Dose-dependent increase in interleukin (IL)-10 protein with BMS-986235 pretreatment (0.03, 0.3, or 3 mg/kg; n = 8 per group) and challenge with lipopolysaccharide. Histological measurements of (B) infarct area, (C) infarct collagen, and (D) infarct matrix metalloproteinase-2 (MMP-2) 74 hours following myocardial infarction (MI) and treatment with vehicle (Veh) or BMS-986235 (3 mg/kg; n = 8/group). (E) Detection and quantification of arginase-1 messenger RNA (mRNA) levels in the peri-infarct zone by in situ hybridization for Veh and BMS-986235 treatment groups (3 mg/kg; n = 4 per group). (F) Representative image of the peri-infarct zone from infarcted mice treated with BMS-986235 (3 mg/kg). Arginase-1 (Arg1) mRNA is indicated in red and FPR2 mRNA is indicated in blue. Data represent the mean ± SEM. Statistical comparisons were done with an unpaired t test or a Dunnett’s test vs Veh when comparing multiple dose groups: ∗P < 0.05, ∗∗∗P < 0.001.

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  Figure 6. Cardiac Inflammation and CD206+ Macrophage Levels Post MI in Mouse Analysis of total leukocytes, neutrophils, and macrophage polarization status in the heart following myocardial infarction (MI) by flow cytometry. (A) Representative bivariate plot of final gate for CD206+ macrophage population. (B) Total CD45+ leukocytes, (C) % of total CD45+ leukocytes that are CD64+ monocyte/macrophages, (D) % of CD64+ monocyte/macrophages that are CD206+ (“M2”), (E) % of CD64+ monocyte/macrophages that are CD206– (“M1”), and (F) % of total CD45+ leukocytes that are Ly6G+ neutrophils. Treatments consisted of vehicle (Veh) (n = 7) or 3 mg/kg BMS-986235 (n = 10). Noninfarcted sham hearts (n = 10) are shown for comparison. Data represent the mean ± SEM. T-test vs Veh: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

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  Figure 7. Effects of BMS-986235 on Post-Infarction Structure-Function in Mouse Treatments were initiated 24 hours after myocardial infarction (MI) and continued to 28 days. Treatments consisted of a suspension vehicle (Veh) and BMS-986235 at either 0.3 mg/kg or 3 mg/kg once per day. (A) Kaplan-Meier plot of post-infarction survival in mice dosed with Veh (n = 24), 0.3 mg/kg (n = 23) BMS-986235, and 3 mg/kg BMS-986235 (n = 23). (B) Representative heart cross-sections by histology depicting the degree of MI. Histomorphometric analysis of (C) scar area, (D) infarct wall thickness, and (E) left ventricular chamber area 28 days after MI. Group sizes: sham (n = 11), Veh (n = 16-17), 0.3 mg/kg BMS-986235 (n = 13-16), and 3 mg/kg BMS-986235 (n = 13-15). Bar graphs show the mean ± SEM. Dunnett’s vs Veh: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Log-rank test to compare survival curves: ∗P < 0.05.

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  Figure 8. Effects of BMS-986235 on Post-Infarction Structure-Function in Rat Treatments were initiated 48 hours after myocardial infarction (MI) and continued to 6 weeks. Treatments consisted of a suspension vehicle (Veh) or BMS-986235 (1 mg/kg) once per day. Endpoints evaluated 6 weeks after MI following permanent coronary artery occlusion: histomorphometric analysis of (A) scar area and (B) infarct wall thickness; echocardiographic measurements of (C) LV end-diastolic volume (EDV) and (D) LV end-systolic volume (ESV); and (E) ejection fraction percentage (EF%). Endpoints evaluated 6 weeks post MI induced via ischemia-reperfusion injury were (F) LV EDV, (G) LV ESV, (H) ex vivo pressure-volume curves, and (I) EF%; (J) histology of heart and LV infarct cross-sections; and (K) regional analysis of viable myocardium across the infarct wall. Group sizes for permanent coronary artery occlusion and ischemia-reperfusion studies: sham (n = 10 for both), Veh (n = 22-25 and n = 14, respectively), and BMS-986235 (n = 20-24 and n = 14, respectively). (F, G, I) Data from indwelling pressure-volume conductance catheter measurements. Data represent the mean ± SEM. Dunnett’s vs Veh: ∗P < 0.05, ∗∗P < 0.01,…