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 (M2EV) 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 M2EV treatment. M2EV rescued cardiac function and attenuated injury markers, infarct size, and scar size. M2EV inhibited CCR2+ macrophage numbers, reduced monocyte‐derived CCR2+ macrophage recruitment to infarct sites, induced M1‐to‐M2 macrophage switching and promoted neovascularization. Analysis of M2EV 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 M2EV actions and resulted in failure to inhibit CCR2+ macrophage numbers and infarct size. Taken together, this investigation showed that M2EV 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.

homology to known gene transcripts (titer: 1.14×10 12 ) were injected into the left ventricle at multiple sites. After a period of 45 days, the rats were subjected to I/R surgery and subsequent treatments. Six-week-old CCR2 CreER/+ R26 tdtomato/+ mice received daily intraperitoneal injections of tamoxifen (2 mg) in corn oil, for 5 days. After three weeks, CCR2 CreER/+ R26 tdtomato/+ mice were prepared for surgery as described above. Seven/eightweek-old CCR2 DTR-GFP mice received daily intraperitoneal injections of DT (50 μg/kg body weight), for 3 days. The following day, CCR2 DTR-GFP mice were prepared for surgery as described above. Seven/eight-week-old Lyz2 Cre/+ R26 tdTomato/+ mice received daily intraperitoneal injections of tamoxifen (2 mg) in corn oil, for 5 days. After 7 days, Lyz2 Cre/+ R26 tdtomato/+ mice were prepared for surgery as described above.

Microarray-based high-throughput microRNA expression profiling:
MicroRNA sequencing was performed using nucleic acids isolated from M2 EV and M1 EV . Analysis and bioinformatics analysis was outsourced and performed by Ouyi Biomedical Technology (Shanghai, China). In short, the Agilent Rat miRNA Microarray Kit (Release 21.0, 8x15K, Design-ID: 070154) was used to evaluate 6 independent samples for the expression profiles of 780 mature miRNA. Total RNA was quantified by a NanoDrop ND-2000 (Thermo-Fisher Scientific) and the RNA integrity (RIN) was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, USA). The sample labeling, microarray hybridization, and washing were performed based on the manufacturer's standard protocols. Briefly, total RNA was dephosphorylated, denaturated, and then labeled with Cyanine-3-CTP. After purification the labeled RNAs were hybridized onto the microarray. After washing, the microarrays were scanned with an Agilent Scanner G2505C (Agilent Technologies). Feature Extraction software (v10.7.1.1, Agilent Technologies) was used to analyze array images to calculate raw data. Next, raw data was normalized using the quantile algorithm. The probes present in at least 100 % of samples in any 1 of the 2 conditions were flagged as "Detected" and were selected for further data analysis. Differentially expressed miRNAs were then identified through fold-change as well as P-values calculated by t-test. The thresholds set for up-and down-regulated genes were a fold-change ≥ 2.0 and a P-value ≤ 0.05.

Cardiac functional measurements:
Rat echocardiography was performed using a Vevo 2100 (VisualSonic, Canada) ultra-high-resolution animal ultrasound imaging system and the left ventricular function was evaluated as previously described. Long axes with M-mode were visualized. Three representative cycles were captured for each animal, and measurements for ejection fraction (EF%), fractional shortening (FS%). Rat left ventricular pressure-volume was determined using a Millar pressure-volume catheter (SPE-869, #840-813G) was connected to an MPVS Ultra PV loop system (Millar, Houston, TX, USA). After calibration, the catheter was advanced into the left ventricle through the right common carotid artery. The pressure-volume loop was recorded using a data acquisition system (LabChart Pro, ADInstruments, Colorado Springs, CO, USA) and measurements for dP/dt max and dP/dt were calculated. Pig echocardiography was performed using a Philips EPIQ 7C ultrasound system (Philips, Cambridge, MA, USA) and was used to evaluate the ejection fraction (EF%) before LAD and after 72 h of reperfusion. Electrocardiograms (ECG, Schiller, AT-102, Switzerland) were recorded before LAD and after 72 h of reperfusion.

2,3,5-Triphenyltetrazolium chloride (TTC) staining:
After 72 h of reperfusion, the rats and pigs were euthanized, hearts were excised, and were washed in PBS. Following harvest, rat hearts were cut into six transverse slices (from apex to basal edge of infarction), whereas pig hearts were sectioned into 1 cm thick slices (below the ligation line). Sections were incubated with 1% 2,3,5-triphenyltetrazolium chloride (TTC) in PBS for 30 min, at 37°C, and in darkness. Then, the transverse slices were washed with PBS three times and subsequently imaged. The infarcted myocardium was stained in white, and the viable tissue was stained in red. Photographs were taken and images were analyzed using ImageJ software (NIH, Bethesda, MD, USA). The percentage of infarct area was calculated using the following equation:

Magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS)
separation ： For single-cell suspensions, the Multi-Tissue Dissociation Kit 2 (Miltenyi, #130-110-203, Germany) was used to dissociate cells from adult rat or mice hearts according to the distributor's recommendations. Briefly, heart tissues harvested from rats and mice were cut at the infarction border zone (~2 mm in width around the infarcted myocardium) into small pieces (1-2 mm³) and suspended in 2.5 mL of enzyme mix in gentleMACS C-Tubes

Quantitative real-time polymerase chain reaction (RT-PCR):
The mRNA reverse transcription kit was used to obtain cDNA following the manufacturer's instructions (Roche, Basle, Switzerland). SYBR Green PCR master mix (Roche) was used to perform RT-PCR using a CFX96TM PCR detection system (BioRad, Redmond, WA, USA). GAPDH and U87 were used as a housekeeping gene. All primer sequences are provided in the supplementary data.     levels of IL-1β, IL-6 and TNF-α in rat heart using ELISA, n=7. 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.  in CCR2 + macrophages, as assessed using Western Blot and densitometric analysis, n=3. (G-H) OCR and ECAR measurements in CCR2 + macrophages, as assessed by a Seahorse Bioscience XF24 analyzer, n=3. 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.

Fig. S8. MicroRNA-181b-5p is implicated in CCR2 regulation by M2 EV . (A)
Representative H&E staining of transverse heart tissue sections. Scale bar = 100 μm. 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.   sections and quantification, n = 3. All 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.