Excessive Neutrophil Extracellular Trap Formation Aggravates Acute Myocardial Infarction Injury in Apolipoprotein E Deficiency Mice via the ROS-Dependent Pathway

Figure 4

ApoE^-/- deficiency regulates NET formation through the NADPH oxidase-ROS-dependent pathway. (a) Representative immunofluorescent staining of cit-H3 (green), Ly6G (red), and DAPI (blue) in ApoE^-/-- and WT-derived neutrophils treated with PMA for 18 hours. Scale bar: 50 μm. (b) Representative live cell staining of SYTOX Green (green, extracellular DNA) and Hoechst 33258 (blue, total DNA) of ApoE^-/-- and WT-derived neutrophils stimulated with PMA for 4 hours. Scale bar: 100 μm. And quantitation of ex vivo NET formation of ApoE^-/-- and WT-derived neutrophils stimulated with PMA for 2 h, 4 h, 6 h, and 18 h by SYTOX Green. (ApoE^-/- mouse-derived neutrophils vs. WT mice 4 hours after stimulation), (ApoE^-/- mouse-derived neutrophils vs. WT mice 6 hours after stimulation), and (ApoE^-/- mouse-derived neutrophils vs. WT mice 18 hours after stimulation). (c) Quantitative real-time PCR of apolipoprotein E (Apoe) in the livers, hearts, and purified neutrophils of WT mice (). (d) Quantitation of ex vivo NET formation of ApoE^-/- mouse- and WT mouse-derived neutrophils stimulated with PMA with or without APOE ε3 (APOE3) supplement for 4 hours. (e) Quantitation of ROS levels of ApoE^-/- mouse- and WT mouse-derived neutrophils stimulated with PMA with or without APOE3 supplement for 4 hours. (f) Quantitation of ex vivo NET formation of ApoE^-/- mouse- and WT mouse-derived neutrophils stimulated with PMA with or without apocynin supplement for 4 hours. (g) Quantitation of ex vivo NET formation of ApoE^-/- mouse- and WT mouse-derived neutrophils stimulated with H₂O₂ with or without APOE3 supplement for 4 hours. Columns filled with black: WT mouse-derived neutrophils. Columns filled with white: ApoE^-/- mouse-derived neutrophils. Data are shown as . Statistical tests include the two-tailed Student’s -test (two groups) and one-way analysis of variance followed by Tukey’s multiple comparison test (more than two groups) or by Dunnett’s multiple comparison test (d, f). and .

(a)

(b)

(c)

(d)

(e)

(f)

(g)