**Background:** Acute myocardial infarction (AMI) triggers a macrophage-mediated inflammatory response that determines cardiac repair or adverse remodeling. Pro-inflammatory CCR2+ macrophages (resembling M1) recruit circulating monocytes and exacerbate injury, while reparative CCR2- macrophages (resembling M2) promote healing. Small extracellular vesicles (sEV) from M2 macrophages (M2~EV~) are hypothesized to mediate anti-inflammatory effects, but their mechanisms on CCR2+ macrophage subpopulations are unclear.
**Methods:** M2~EV~ were isolated from IL-4/IL-13-polarized rat bone marrow-derived macrophages (BMDM) by ultracentrifugation and characterized by TEM, NanoFCM, and Western blot (Alix, CD9, CD63, calnexin). Rat and porcine I/R models were created by 60-min LAD ligation followed by reperfusion. M2~EV~ (rat: 150 µg; pig: 1.0 mg) or controls (saline, M1~EV~) were injected intramyocardially 30 min post-reperfusion. Cardiac function was assessed by echocardiography (LVEF, LVFS) and pressure-volume loops (dP/dt). Infarct size was measured by TTC staining. Serum LDH, CK, CK-MB (rats) and cardiac troponin (pigs) were measured. Macrophage subpopulations were analyzed by flow cytometry (MHC-II/CCR2), immunostaining (CD68, CCR2, ARG-1, IL-1β), and fate mapping using CCR2^CreER/+^ R26^tdTomato/+^ mice to distinguish monocyte-derived vs. tissue-resident CCR2+ macrophages. Gene expression (RT-qPCR) and protein levels (Western blot, ELISA) of inflammatory and repair markers were assessed. Metabolic assays (Seahorse OCR/ECAR, glucose uptake via 2-NBDG, mtROS via MitoSOX) were performed on sorted CCR2+ macrophages. miRNA profiling identified miR-181b-5p as a candidate; its role was tested using lentiviral inhibition (M2~EV-i181~).
**Key Results:** In rats, M2~EV~ significantly improved LVEF (from ~38% to ~55%, p<0.01) and LVFS, increased dP/dt max, reduced dP/dt min, and decreased serum LDH, CK, and CK-MB (all p<0.01 vs. I/R). Infarct size was reduced from 38.1% (I/R) to 18.7% (M2~EV~) vs. 31.6% (M1~EV~). In pigs, M2~EV~ improved LVEF (p<0.01 vs. I/R), reduced infarct size, and lowered cardiac troponin at 48 and 72 h (p<0.05). M2~EV~ reduced MHC-II^Hi^/CCR2+ macrophage proportions (I/R: 25.1±2.8%; M2~EV~: 10.6±4.0%; p<0.01) and decreased both monocyte-derived and tissue-resident CCR2+ macrophages in fate-mapping mice. M2~EV~ increased ARG-1+ reparative macrophages and decreased IL-1β+ pro-inflammatory macrophages. In vitro, M2~EV~ inhibited LPS+IFNγ-induced NFκB nuclear translocation and promoted M2 polarization. M2~EV~ reduced glucose uptake and mtROS in CCR2+ macrophages and shifted metabolism away from glycolysis (reduced ECAR). miRNA profiling revealed 55 upregulated miRNAs in M2~EV~ vs. M1~EV~; miR-181b-5p was selected. Functional blockade of miR-181b-5p (M2~EV-i181~) abolished the reduction in CCR2+ macrophages, failed to improve LVEF/LVFS, and did not reduce infarct size or serum injury markers. M2~EV-i181~ also failed to reduce glucose uptake, mtROS, or glycolysis in CCR2+ macrophages, and did not suppress STAT3 protein expression.
**Clinical Implications:** This study demonstrates that M2~EV~, via miR-181b-5p, regulate CCR2+ macrophage metabolism and polarization, reducing inflammation and promoting cardiac repair after AMI. The findings support the development of cell-free sEV-based therapies for myocardial I/R injury, potentially avoiding limitations of cell transplantation. However, challenges remain, including the need for autologous sEV sources, sufficient yield, and optimal delivery routes (e.g., intrapericardial injection with hydrogels). The results also highlight the importance of targeting CCR2+ macrophages and glucose metabolism in post-AMI inflammation.