Aminooxyacetic acid attenuates post‐infarct cardiac dysfunction by balancing macrophage polarization through modulating macrophage metabolism in mice

Figure 1. Aminooxyacetic acid (AOAA) inhibits glycolysis and enhances ATP levels in classically activated M1 macrophages. (A) FACS analysis of the macrophage surface marker CD11b and F4/80 of BMDMs. (B) pH values of BMDMs culture media, unstimulated (M0) or stimulated with LPS/INF‐γ ± AOAA (1, 5 mmol/L). (C) Lactic acid production of BMDMs after stimulation with LPS/INF‐γ ± AOAA (1, 5 mmol/L). (D) ATP level of BMDMs measured using ATP assay kit after stimulation with LPS/INF‐γ ± AOAA (1, 5 mmol/L). (E) Measurement of ECAR of BMDMs unstimulated (M0) or stimulated with LPS/INF‐γ ± AOAA (1, 5 mmol/L). (F) ECAR of BMDMs unstimulated (M0) or stimulated with LPS/INF‐γ ± AOAA (1, 5 mmol/L). (G) Gene expression profiles of limiting enzymes in glucose catabolism (HK2, PFKL, PKM2, GFAT1, 6PGDH and G6PDH) in BMDMs after stimulation with LPS/INF‐γ ± AOAA (1, 5 mmol/L). ns: no significant difference, *P < .05, **P < .01. Data are represented as mean ± SEM. HBP, hexosamine biosynthesis pathway; PPP, pentose phosphate pathway

Figure 2. Aminooxyacetic acid (AOAA) blocks LPS/INF‐γ induced classically activated M1 macrophages phenotype and pro‐inflammatory cytokines expression in vitro. (A) Representative FACS analysis of the M1 macrophages surface marker CD86 of BMDMs and the percentages of CD86 positive cells, unstimulated (M0) or stimulated with LPS/INF‐γ ± AOAA (1, 5 mmol/L). (B) Representative confocal fluorescence imaging of iNOS in BMDMs after incubating with LPS/INF‐γ ± AOAA (1, 5 mmol/L). Scale bar equals 40 μm. (C) Quantification of iNOS Fluorescence intensity. (D) Representative immunoblot band of iNOS in BMDMs after stimulation with LPS/INF‐γ ± AOAA (1, 5 mmol/L). (E) Gene expression profiles of iNOS, IL‐6 and TNF‐α in BMDMs after stimulation with LPS/INF‐γ ± AOAA (1, 5 mmol/L). ns: no significant difference *P < .05, **P < .01. Data are represented as mean ± SEM

Figure 3. Aminooxyacetic acid (AOAA) promotes alternative active M2 macrophages polarization and anti‐inflammatory cytokine expression in vitro. (A) Representative confocal fluorescence imaging of Arg1 in BMDMs after incubation with IL‐4 ± AOAA (1, 5 mmol/L). Scale bar equals 40 μm. (B) Quantification of Arg1 Fluorescence intensity. (C) Representative immunoblot band of Arg1 in BMDMs after stimulation with IL‐4 ± AOAA (1, 5 mmol/L). (D) Gene expression profiles of Arg1, CD206 and IL‐10 in BMDMs after stimulation with IL‐4 ± AOAA (1, 5 mmol/L). ns: no significant difference, *P < .05, **P < .01. Data are represented as mean ± SEM

Figure 4. Aminooxyacetic acid (AOAA) administration attenuates post‐myocardial infarction (MI) cardiac dysfunction and infarct size in mice. (A) Ejection fraction and (B) fractional shortening of saline‐treated MI mice and AOAA‐treated MI mice were measured by echocardiography at baseline, day 3, day 7, day 14 and day 28 after MI surgery. (C) Representative cross‐sectional images of hearts stained with Masson's trichrome at day 28 post MI surgery. Serial sectioning was performed at 500 μm intervals. Scale bar equals 2 mm. Blue, scar tissue; red, viable myocardium. (D) Quantitative of the infarct percentage in heart sections following MI analysed by Masson's trichrome staining. *P < .05, Data are represented as mean ± SEM

Figure 5. Aminooxyacetic acid (AOAA) reduces the proportion of M1 macrophages and boosts M2‐like phenotype in the ischaemic border zone. (A) Representative immunofluorescence double staining of F4/80 (red) and iNOS (green) or Arg1 (green) within the hearts of mice treated with saline or AOAA for 3 days after myocardial infarction (MI). Scale bar equals 20 μm. (B) Quantification of total macrophages (F4/80+), M1 macrophages (F4/80+iNOS+) and M2 macrophages (F4/80+Arg1+) within the hearts of mice treated with saline or AOAA for 3 days after MI. (C) Representative images of Western blot of iNOS and Arg1 in the hearts of saline‐treated MI mice and AOAA‐treated MI mice. (D) Gene expression profiles of iNOS and Arg1 in the hearts of mice treated with saline or AOAA for 3 days after MI. ns: no significant difference, *P < .05, **P < .01. Data are represented as mean ± SEM

Figure 6. Aminooxyacetic acid (AOAA) treatment blunts cardiac inflammation response in mice with myocardial infarction (MI). (A) Phothmicrograph of representative HE staining sections and quantification of inflammatory cell infiltration (%) within the heart of mice treated with saline or AOAA for 3 days after MI. Scale bar equals 100 μm. (B) Representative confocal fluorescence imaging of TNF‐α within the hearts of mice treated with saline or AOAA for 3 days after MI. Scale bar equals 10 μm. (C) Quantification of TNF‐α positive cells. (D) Representative confocal fluorescence imaging of CD206 within the hearts of mice treated with saline or AOAA for 3 days after MI. Scale bar equals 10 μm. (E) Quantification of CD206 positive cells. (F) Gene expression profiles of TNF‐α, IL‐6 and IL‐10 in the hearts of mice treated with saline or AOAA for 3 days after MI. *P < .05, **P < .01. Data are represented as mean ± SEM

Figure 7. Aminooxyacetic acid (AOAA) administration attenuates NLRP3‐Caspase1/IL‐1β activation in both ischaemic myocardium and LPS/INF‐γ‐stimulated BMDMs. (A) Representative images of Western blot of NLRP3 and Caspase1 in the hearts of mice treated with saline or AOAA for 3 days after myocardial infarction (MI). (B) Gene expression profile of IL‐1β in the hearts of mice treated with saline or AOAA for 3 days after MI. (C) Representative images of Western blot of NLRP3 and Caspase1 in BMDMs after stimulation with LPS/INF‐γ ± AOAA (1, 5 mmol/L). (D) Gene expression profile of IL‐1β in BMDMs after stimulation with LPS/INF‐γ ± AOAA (1, 5 mmol/L). **P < .01. Data are represented as mean ± SEM

Figure 8. A schematic illustration on possible roles of aminooxyacetic acid (AOAA) on macrophage metabolism and polarization. Two break points lie in TCA of M1 macrophages. Aspartate‐arginosuccinate shunt compensates them. AOAA hinders succinate accumulation by inhibiting AST, resulting in the transcription inhibition of glycolysis and pro‐inflammation genes and the inhibition of ROS generating. 6PGDH, 6‐phosphogluconate dehydrogenase; AST, aspartate aminotransferase; F1,6BP, fructose‐1,6 diphosphate; F6P, fructose‐6‐phosphate; G6P, glucose‐6‐phosphate; G6PDH, glucose 6‐phosphate dehydrogenase; GFAT1, glutamine: fructose‐6‐phosphate aminotransferase 1; HBP, hexosamine biosynthetic pathway; HK, hexokinase; IDH, isocitrate dehydrogenase; NO, nitric oxide; OAA, oxaloacetate; OXPHOS, oxidative phosphorylation; PEP, phosphoenolpyruvate; PFKL, phosphofructokinase liver; PKM2, pyruvate kinase M2; PPP, pentose phosphate pathway; ROS, reactive oxygen species; SDH, succinate dehydrogenase; TCA, tricarboxylic acid cycle; α‐KG, α‐ketoglutarate