M2 Macrophage-Derived sEV Regulate Pro-Inflammatory CCR2+ Macrophage Subpopulations to Favor Post-AMI Cardiac Repair

Abstract

Tissue-resident cardiac macrophage subsets mediate cardiac tissue inflammation and repair after acute myocardial infarction (AMI). CC chemokine receptor 2 (CCR2)-expressing macrophages have phenotypical similarities to M1-polarized macrophages, are pro-inflammatory, and recruit CCR2⁺ circulating monocytes to infarcted myocardium. Small extracellular vesicles (sEV) from CCR2^̶ macrophages, which phenotypically resemble M2-polarized macrophages, promote anti-inflammatory activity and cardiac repair. Here, the authors harvested M2 macrophage-derived sEV (M2_EV ) from M2-polarized bone-marrow-derived macrophages for intramyocardial injection and recapitulation of sEV-mediated anti-inflammatory activity in ischemic-reperfusion (I/R) injured hearts. Rats and pigs received sham surgery; I/R without treatment; or I/R with autologous M2_EV treatment. M2_EV rescued cardiac function and attenuated injury markers, infarct size, and scar size. M2_EV inhibited CCR2⁺ macrophage numbers, reduced monocyte-derived CCR2⁺ macrophage recruitment to infarct sites, induced M1-to-M2 macrophage switching and promoted neovascularization. Analysis of M2_EV microRNA content revealed abundant miR-181b-5p, which regulated macrophage glucose uptake, glycolysis, and mitigated mitochondrial reactive oxygen species generation. Functional blockade of miR-181b-5p is detrimental to beneficial M2_EV actions and resulted in failure to inhibit CCR2⁺ macrophage numbers and infarct size. Taken together, this investigation showed that M2_EV rescued myocardial function, improved myocardial repair, and regulated CCR2⁺ macrophages via miR-181b-5p-dependent mechanisms, indicating an option for cell-free therapy for AMI.

Keywords: CC chemokine receptor 2; extracellular vesicles; ischemia-reperfusion injury; macrophage metabolic reprogramming; macrophages.

Figure 1

Protective activity of intramyocardially administered M2_EV in I/R rat hearts. A) Schematic overview of the experimental procedure. Quantification of B) LVEF % and C) LVFS % 3 days after I/R, data are presented as mean ± SD of n = 10 rats. D,E) Quantification of maximal and minimal LV pressure derivative (dP/dt), data are presented as mean ± SD of n = 7 rats. F–H) Blood serum analyses of LDH, CK, and CK‐MB enzyme levels, respectively, data are presented as mean ± SD of n = 10 rats. I,J) Representative TTC‐stained heart images and quantified IS % (rat hearts were cut into six transverse slices (from apex to basal edge of infarction) 3 days after I/R, data are presented as mean ± SD of n = 5 rats. K) Representative H&E staining of heart transverse sections (scale bar = 100 µm) 3 days after I/R. Statistical significance is indicated as **p < 0.01 compared with the sham group and ^## p < 0.01/^# p < 0.05 compared with the I/R group.

Figure 2

M2EV demonstrates a cardioprotective effect against I/R injury in porcine models. A) Schematic of the experimental procedure. B) ECG readings pre‐I/R and at experimental endpoints. C) Representative M‐mode echocardiography images. D) Quantification of LVEF %. E) TTC staining of transverse heart sections to visualize the IS area in pig hearts. F) IS quantification. G) Representative H&E staining of healthy tissue, the area at the risk border zone, and the IS area (scale bar = 50 µm). H) Levels of cTn (ng mL⁻¹) measured in pig plasma at the indicated stages of the experimental procedure. All data are presented as mean ± SD of n = 3 pigs. Statistical significance is shown as ^## p < 0.01 and ^# p < 0.05 compared with the I/R group.

Figure 3

M2_EV inhibits CCR2⁺ macrophage presence in I/R hearts. A) Schematic of the experimental approach. B,C) Flow cytometry analysis, and quantification of MHC‐II^Hi/CCR2⁺ cells, respectively, n = 4. D) Representative immunostaining of CD68, CCR2, ARG‐1, and IL‐1β. E,F) Quantitative analysis of CCR2⁺ IL‐1β ⁺ ARG‐1⁻, and CCR2^−/+ IL‐1β ⁻ ARG‐1⁺ macrophages from stained tissue sections in D (n = 3 rats in each group, three microscopic fields of view for each sample, scale bar = 20 µm). G) Experimental design for tamoxifen‐treated CCR2^CreER/+ R26^tdtomato/+ mice to deplete CCR2⁺ circulating monocytes. H,I) Quantification of resident and monocyte‐derived cardiac CCR2⁺ macrophages, hearts from 6–8 mice were mixed as one sample, n = 3. J–P) Phenotyping of tissue‐resident and monocyte‐derived cardiac CCR2⁺ macrophages using RT‐PCR to assess gene expression from fate mapping experiments outlined in G, n = 4. Q–U) The protein levels of matrix metalloproteinase 9 (MMP9), transforming growth factor (TGF)‐β, vascular endothelial growth factor A (VEGFA), and Mertk in tissue‐resident and monocyte‐derived cardiac CCR2⁺ macrophages using Western blot, n = 3. V–X) The protein levels of IL‐1β, IL‐6, and tumor necrosis factor (TNF)‐α in tissue‐resident and monocyte‐derived cardiac CCR2⁺ macrophages using ELISA, n = 4. All data are presented as the mean ± SD. Statistical significance is shown as **p < 0.01 compared with the sham group and ^## p < 0.01 compared with the I/R group.

Figure 4

Mitochondrial respiration and glycolysis assays in CCR2⁺ macrophages. A,B) OCR and C,D) ECAR measurements in cardiac tissue‐resident and monocyte‐derived CCR2⁺ macrophages, as measured using a Seahorse Bioscience XF24 analyzer, n = 3, probed by the serial addition of A: oligomycin, B: FCCP, and C: antimycin A/rotenone as indicated. E) Glucose uptake in monocyte‐derived and locally resident CCR2⁺ macrophages measured using the fluorescence‐labeled glucose analogue (2‐NBDG) by mean fluorescence intensity (MFI), n = 6. F) mtROS levels in monocyte‐derived and locally resident CCR2⁺ macrophages using MitoSOX fluorescent probe, n = 6. All data are presented as the mean ± SD. Statistical significance is shown as ^**p < 0.01 compared with the sham group and ^##p < 0.01 compared with the I/R group.

Figure 5

M2_EV inhibition of the CCR2 macrophage population promotes angiogenesis in rat and porcine myocardial I/R injury models. Co‐immunostaining of A) CCR2/CD31, CCR2/VEGF, and CCR2/α‐SMA in tissue sections of the infarction border zone of rat hearts. B–D) Quantitative analysis of CCR2: marker ratios from stained tissue sections in A (n = 3 rats in each group, 3–5 microscopic fields of view for each sample, scale bar = 100 µm). E) Immunostaining of α‐SMA in area at the risk and infarct areas of porcine hearts. F) Quantitative analysis of α‐SMA: marker ratios from stained tissue sections in E (n = 3 pigs per group, 3–5 microscopic fields of view for each sample, scale bar = 100 µm). Co‐immunostaining of G) CD31/BrdU and H) α‐SMA/BrdU in tissue sections of the infarction border zone of rat hearts (n = 3 rats in each group, scale bar = 50 µm, white arrows indicate positively co‐stained cells). Data presented as the mean ± SD. Statistical significance is shown as *p < 0.05 and ^** p < 0.01 compared with the control group; ^# p < 0.05 and ^## p < 0.01 compared with the I/R group.

Figure 6

MicroRNA‐181b‐5p is implicated in CCR2 regulation by M2_EV. A) Flow cytometry analysis of MHC‐II^Hi/CCR2⁻ and MHC‐II^Hi/CCR2⁺ cells in the infarct border zone tissue of rat I/R injured hearts. B) Quantified changes in MHC‐II^Hi/CCR2⁺ cell populations under each treatment condition, n = 4. C) CCR2 mRNA expression in CCR2‐lentivirus infected BMDM incubated with M2_EV or M2_EV‐i181, n = 3. D,E) Quantification of LVEF and LVFS, n = 6. F–H) Levels of LDH, CK, and CK‐MB in blood serum, n = 6. I,J) OCR and ECAR measurements in CCR2⁺ macrophages, as assessed by a Seahorse Bioscience XF24 analyzer, n = 3, probed by the serial addition of A: oligomycin, B: FCCP, and C: antimycin A/rotenone as indicated. K) Quantified mtROS MFI in CCR2⁺ macrophages, n = 6. L) Glucose uptake in CCR2⁺ macrophages measured using the fluorescence‐labeled glucose analogue, 2‐NBDG, n = 6. M–P) Phenotyping of resident and monocyte‐derived cardiac CCR2⁺ macrophages with M2_EV or M2_EV‐i181, n = 4. Q,R) The protein levels of IL‐1β and IL‐6 in tissue‐resident and monocyte‐derived cardiac CCR2⁺ macrophages, as measured using ELISA, n = 4. S–U) The protein levels of MMP9 and VEGFA in tissue‐resident and monocyte‐derived cardiac CCR2⁺ macrophages, as detected using Western blot, n = 3. V) Co‐immunostaining of CCR2 and α‐SMA, and Sirius Red staining of collagen fibrils. Scale bar = 100 µm. W) Quantitative analysis of α‐SMA and CCR2 from Q (three rats in each group, 3–5 microscopic fields of view for each rat, scale bar = 100 µm). Data are presented as the mean ± SD. Statistical significance is shown as *p < 0.05 and ^** p < 0.01 compared with the control group; ^# p < 0.05 and ^## p < 0.01 compared with the I/R group.