Figure 3.

KLF2-EVs reduced inflammation level after I/R injury by decreasing Ly6C^high monocytes. (A) Representative immunofluorescence staining of Ly6C^high phenotype (F4/80+iNOS+) and Ly6C^low phenotype (F4/80+CD163+) in hearts of sham-operated, PBS or KLF2-EVs treated mice and quantification of double positive cells proportion in F4/80+ cells (B, C) 3 days following I/R injury (n=5). Scale bar= 50 µm. (D) Representative flow cytometry plots showing Ly6C^high monocyte/macrophage (Mo/Mø) (CD11b+Ly6C^high) and Ly6C^low Mo/Mø (CD11b+Ly6C^low), and quantification of cells within heart tissue 3 days following treatment (n=6). (E) Representative flow cytometry plots showing total monocytes, Ly6C^high monocytes (CD11b+Ly6C^high) and Ly6C^low monocytes (CD11b+Ly6C^low), and quantification of cells in peripheral blood 3 days following treatment (n=6). (F) Schematic of Mo/Mø depletion protocol with Cl₂MDP liposomes. (G) Ejection fraction (EF) and left ventricular end-diastolic diameter (LVDd) of sham-operated, PBS or KLF2-EVs treated mice measured by echocardiography 3 days after myocardial I/R injury combined with systemic depletion of endogenous Mo/Mø (n=5). (H) Representative flow cytometry plots showing apoptosis of monocytes, primary gate is Ly6C^high Mo/Mø (CD11b+Ly6C^high) or Ly6C^low Mo/Mø (CD11b+Ly6C^low) in heart tissue 3 days following treatment, and quantification of Annexin V+ cells proportion in primary gate (n=5). Graphs depict mean ± SD. Statistical significance was measured via one-way ANOVA followed by Tukey's multiple comparisons test for multiple groups' comparison and two-way ANOVA followed by Bonferroni's multiple comparisons test for comparison between different groups in different cell subtypes. **P < 0.01, ****P < 0.0001, ns= not significant.