| BackgroundAcute myocardial infarction(AMI)is one of the major causes of death worldwide.Within the development of coronary interventional therapy,the majority of patients with AMI are survived because the dying myocardium is saved through coronary revascularization.However,the process of reperfusion itself could induce further damage known as myocardial ischemia/reperfusion injury(myocardial I/R injury),manifested as myocardial stunning,arrhythmia and changes in myocardial energy metabolism.Myocardial I/R injury has become the main obstacle for patients to get benefits from coronary revascularization.How to prevent and reduce myocardial I/R injury is still an unsolved clinical problem.Immune activation plays an important role in the occurrence and development of myocardial I/R injury.Upon reperfusion,the restored blood flow reintroduces oxygen,leading to the generation of damaging oxygen free radicals.Due to the large amount of oxygen free radicals,the stressed cardiomyoctes release endogenous damage-associated molecular patterns(DAMPs),which recruit inflammatory cells to injured myocardium,triggering and amplifying local inflmmatrory response and resulting in secondary damage to cardiomyotes.Monocyte/macrophages play an important role in the inflammatory response triggerd by myocardial I/R injury.Initially,M1 macrophages are in dominant and release a variety of pro-inflammatory cytokines and chemokines,thus generating a pro-inflammatory environment and removing cell debris.With the passage of time,the negative effects of M1 macrophages on tissue damage and cardiomyoctes apoptosis gradually exacerbated,macrophages gradually transformed into M2 type.The M2 macrophages secrete not only anti-inflammatory cytokines but also a mixture of growth factors and play a critical role in wound healing and scar formation.Hence,How to promote the transformation of M1 macrophage to M2 type after myocardial I/R injury has received more and more attention.Mesenchymal stem cells(MSCs)are a unique adult stem cell type with self-renewal and multi-directional differentiation potential,which are widely used in the regeneration and repair of injured myocardium.Previous studies demonstrated that MSC could differentiate into cardiomyocyte-like cells after transplanted into I/R injured myocardium.However,with the deepening of research,accumulating evidence indicated that MSC exerted its benefit on myocardium via its paracrine effect rather than differentiating into myocytes.Exosomes are membrane vesicles,secreted by a large variety of cells,with diameters of 30-150 nm,and contain cell-specific proteins,lipids and nucleic acids.At present,exosomes are considered as an important basis for MSC paracrine activity.This study aims at investigating whether MSC-derived exosomes play a protective role in myocardial I/R injury by regulating the polarization of macrophages and exploring the possible molecular mechanisms involved in it,thus providing theoretical basis for its application in clinical trials.Materials and Methods(1)Exosomes were purified from the supernatant of MSCs using gradient centrifugation method,and identified by nanoparticle trafficking analysis(NTA),transmission electron microscopy and western blotting.(2)Mice underwent myocardial ischemia/reperfusion were infused with MSC derived exosomes(MSC-Exo)or vehicle immediately after reperfusion through intramyocardial injection.The curative effects of MSC-Exo on myocardial ischemia/reperfusion and its following cardiac inflammation were evaluated.(3)We tested whether systemic depletion of macrophages would recapitulate the benefits of MSC-Exo therapy by injecting clodronate liposomes into the tail vein of mice.(4)The modifying effects of MSC-Exo on macrophage phenotype were evaluated in vivo and in vitro by flow cytometry and immunoblot analysis.(5)According to miRNA sequencing results of MSC-Exo and previous literature,miRNAs,which might be involved in macrophage phenotype modulation,were screened and further verified by miRNA functional experiments.(6)The downstream pathways were explored by immunoblot analysis and other experiments based on the bio informatics prediction of miRNA target gene.Results(1)Exosomes with lipid bilayer membrane structure,within 30-150nm in diameter expressing specific surface marker were successfully isolated from culture medium using ultracentrifugation method(2)Intramyocardial injection of MSC exosomes immediately after reperfusion could improve cardiac function,reduce infarct size,inhibit ventricular remodeling and attenuate cardiac inflammation.(3)Systemic depletion of macrophages impaired the benefits of MSC-Exo,indicating that macrophages were required for the cardioprotective effects of MSC-Exo therapy.(4)MSC-Exo polarized macrophages away from the M1 phenotype and toward an M2-like state in vivo and vitro(5)miR-182,enriched in MSC-Exo,were proved to promote the conversion of M1 macrophages to M2 macrophages,while inhibiting miR-182 in MSC-Exo blunted those effects.(6)miR-182 shuttling by MSC-Exo modulated macrophage polarization through inhibiting TLR4/NF-κB signaling pathways and activating PI3K/Akt signaling pathways.ConclusionsIn summary,MSC derived exosomes attenuate myocardial I/R injury and reduce cardiac inflammation via shifting the polarization state of macrophage toward M2 phenotype within the heart.miR-182 packaged in MSC derived exosomes is involved in the modulation of macrophage polarization by targeting TLR4/NF-KB/PI3K/Akt signaling cascades. |
