Exosomal miR-27b-3p Derived from Hypoxic Cardiac Microvascular Endothelial Cells Alleviates Rat Myocardial Ischemia/Reperfusion Injury through Inhibiting Oxidative Stress-Induced Pyroptosis via Foxo1/GSDMD Signaling.

Exosomes derived from cardiac microvascular endothelial cells (CMECs) under hypoxia can mediate cardiac repair functions and alleviate pyroptosis and oxidative stress during ischemia-reperfusion (I/R) injury. This study is aimed at investigating the effect and mechanism of miR-27b-3p underlying hypoxic CMECs-derived exosomes against I/R injury. CMECs were isolated from the left ventricle of Sprague-Dawley rats, followed by culturing under hypoxic conditions or pretreatment with the miR-27b-3p inhibitor. CMECs-derived exosomes were added into H9C2 cells before hypoxia/reoxygenation (H/R) or injected into the rat heart before I/R injury. An in vivo I/R injury model was established by ligating and releasing the left anterior descending coronary artery. Expression of pyroptosis-related factors was detected using Western blot, and heart infarcted size was determined by the 2,3,5-triphenyl-2H-tetrazpinolium chloride staining method. Dual-Luciferase Reporter assays were performed to analyze the interactions of nmiR-27b-3p-forkhead box O1 (Foxo1) and Gasdermin D- (GSDMD-) Foxo1. Chromatin-immunoprecipitation (ChIP) assays were performed to validate the interactions between forkhead box O1 (Foxo1) and Gasdermin D (GSDMD) and Foxo1-mediated histone acetylation of GSDMD. CMECs were successfully identified from left ventricle of Sprague-Dawley rats. The expressions of Foxo1 and pyroptosis-related proteins (GSDMD, NLPR3, cleaved caspase 1, IL-1β, and IL-18) were upregulated in the rat heart after I/R injury. Treatment of CMEC-derived exosomes, especially that under hypoxic conditions, significantly reduced pyroptosis in the rat heart. miR-27b-3p was significantly upregulated in CMEC-derived exosomes under hypoxic conditions, and miR-27b-3p inhibition in exosomes alleviated its cytoprotection and inhibited oxidative stress in H9C2 cells. Treatment with Foxo1 overexpression plasmids aggravated in vitro H/R and in vivo I/R injury by upregulating pyroptosis-related proteins. Further experiments validated that miR-27b-3p negatively targeted Foxo1, which bound to the promoter region of GSDMD. These results demonstrated a great therapeutic efficacy of miR-27b-3p overexpression in hypoxic CMEC-derived exosomes in preventing the development of myocardial damage post I/R injury through inhibiting Foxo1/GSDMD signaling-induced oxidative stress and pyroptosis.

Zhang B ，Sun C ，Liu Y ，Bai F ，Tu T ，Liu Q ... -  《-》

Hypoxia-elicited cardiac microvascular endothelial cell-derived exosomal miR-210-3p alleviate hypoxia/reoxygenation-induced myocardial cell injury through inhibiting transferrin receptor 1-mediated ferroptosis.

Ferroptosis is a novel mode of non-apoptotic cell death induced by build-up of toxic lipid peroxides (lipid-ROS) in an iron dependent manner, which is a key event in ischemia/reperfusion (I/R)-induced cardiomyocytes damages. Studies indicated that ischemic preconditioning with cardiac microvascular endothelial cells (CMECs) protected against I/R-induced cardiomyocytes damages. However, the role of hypoxia-conditioned CMECs-derived Exo (H-exo) in I/R cardiomyocytes damages remains largely unclear. Therefore, the objective of this study was to explore the role and underlying mechanisms of H-exo in hypoxia/reoxygenation(H/R)-induced H9C2 cells damages. The rat CMECs were subjected to hypoxia or normoxia culture and Exo was subsequently collected and identified. H-exo or normoxia-conditioned CMECs-derived Exo (N-exo) were administered to H9C2 cells with H/R. To evaluate the therapeutic effect of H-exo and H-exo on H/R-induced H9C2 cells damages, cell proliferation was detected by CCK-8 assay and Edu staining, and ferroptosis process were evaluated by iron ion concentration, lipid reactive oxygen species (ROS) level, malondialdehyde (MDA) level, glutathione peroxidase (GSH-Px) level, and the protein expression of ferroptosis markers. Mechanically, we utilized the RT-qPCR to identify the expression of candidate miR-210-3p in N-exo and H-exo. Bioinformatics combined with dual luciferase reporter assay disclosed the downstream molecular mechanism of miR-210-3p. The results indicated that both H-exo and N-exo significantly facilitated cell proliferation, increased GSH-Px levels and ferroptosis marker (GPX4) protein levels, and reduced iron ion concentration, lipid ROS level, MDA levels and ferroptosis markers (ACSL4 and PTGS2) protein levels in H/R-treated H9C2 cells. More importantly, the therapeutic effect of H-exo was significantly better than that of N-exo. Mechanistically, the results of RT-qPCR revealed significant enrichment of miR-210-3p in H-exo compared with N-exo. The miR-210-3p delivered by H-exo inhibited TFR expression by directly interacting with TFR mRNA, resulting in the promotion of cell proliferation and the attenuation of cell ferroptosis in H/R-treated H9C2 cells. All these data demonstrated that H-exo derived miR-210-3p facilitated the proliferation of myocardial cells in H/R-treated H9C2 cells by suppressing TFR-mediated ferroptosis, which provided new methods to treat H/R-induced myocardial injury.

Lei D ，Li B ，Isa Z ，Ma X ，Zhang B ... -  《-》

Retracted: Exosomal miR-27b-3p Derived from Hypoxic Cardiac Microvascular Endothelial Cells Alleviates Rat Myocardial Ischemia/Reperfusion Injury through Inhibiting Oxidative Stress-Induced Pyroptosis via Foxo1/GSDMD Signaling.

Longevity OMAC 《-》

Bone marrow stromal cell-derived exosomes improve oxidative stress and pyroptosis in doxorubicin-induced myocardial injury in vitro by regulating the transcription of GSDMD through the PI3K-AKT-Foxo1 pathway.

Doxorubicin (DOX) can contribute to severe myocardial injury, and bone marrow stromal cells (BMSC)-exosomes (Exos) improves acute myocardial infarction. Hence, this research investigated whether BMSC-Exos alleviated DOX-induced myocardial injury. BMSC-derived Exos were isolated and identified, and the optimal concentration of DOX was confirmed. H9C2 cells were treated with DOX and BMSC-Exos or in combination with the protein kinase B (AKT) inhibitor. Reactive oxygen species (ROS) and JC-1 were detected to assess oxidative stress (OS) and mitochondrial membrane damage, respectively. In addition, the expression of pyroptosis-related molecules was measured. The expression of phosphatidylinositol 3 kinase (PI3K)-AKT pathway-related proteins and the phosphorylation and acetylation of forkhead box O1 (Foxo1) in the cell nucleus and cytoplasm were tested. Last, interactions between Foxo1 and gasdermin D (GSDMD) were assessed. BMSC-Exo treatment increased viability and mitochondrial membrane potential and reduced lactic dehydrogenase release and ROS levels in DOX-treated H9C2 cells. Furthermore, the addition of BMSC-Exos suppressed DOX-induced activation and upregulation of NLRP3 and apoptosis-associated speck-like protein containing A CARD (ASC) and in vitro cleavage of caspase-1, GSDMD, interleukin (IL)-1β, and IL-18 proteins. Additionally, BMSC-Exo treatment enhanced the expression of phosphorylated (p)-PI3K, p-AKT, and p-mTOR in DOX-treated H9C2 cells and the levels of phosphorylated Foxo1 in the cytoplasm of DOX-treated H9C2 cells. Foxo1 was enriched in the promoter region of GSDMD. Moreover, the AKT inhibitor API-2 annulled the effects of BMSC-Exos on OS, pyroptosis, and Foxo1 phosphorylation in DOX-treated H9C2 cells. BMSC-Exos phosphorylated Foxo1 and inactivated Foxo1 transcription via the PI3K-AKT pathway to diminish GSDMD expression, thus restraining DOX-induced pyroptosis and OS of myocardial cells.

Zeng H ，Yang Y ，Tou F ，Zhan Y ，Liu S ，Zou P ，Chen Y ，Shao L ... -  《Immunity Inflammation and Disease》

MiR-183-5p overexpression in bone mesenchymal stem cell-derived exosomes protects against myocardial ischemia/reperfusion injury by targeting FOXO1.

Exosomes have been suggested to serve as possible drug delivery vehicles due to their nanometer-size range and capability of transferring biological materials to recipient cells. Thus, whether miR-183-5p-overexpressing bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) could protect against myocardial ischemia/reperfusion (MI/R) injury by targeting FOXO1 was investigated. Exosomes were isolated from rat BMSCs, and ischemia/reperfusion (I/R) rat models were established. I/R rats were treated with Exo/NC-Exo/miR-183-5p-Exo/anti-miR-183-5p-Exo. Cardiac function, serum biochemical indices, apoptosis, myocardial infarction size, and the expression of miR-183-5p, FOXO1 and cleaved caspase 3 were assessed. Primary cardiomyocytes were isolated to establish hypoxia/reoxygenation (H/R) models to observe the function of miR-183-5p-Exo in vitro. Rats in the I/R group exhibited a decreased left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS) and left ventricular systolic pressure (LVSP) but an increased left ventricular end-diastolic pressure (LVEDP), myocardial infarct size and apoptosis index (AI). In addition, in I/R rats, miR-183-5p expression was decreased, but FOXO1 and cleaved caspase 3 expression was increased. Both Exo and miR-183-5p-Exo improved the above indices in I/R rats, but miR-183-5p-Exo showed better effects. However, anti-miR-183-5p-Exo reversed the protective effect of Exo. FOXO1 was a target gene of miR-183-5p. Experiments in vitro revealed that Exo and miR-183-5p-Exo suppressed apoptosis and oxidative stress injury in H/R-induced cardiomyocytes, whereas overexpressed FOXO1 reversed the protective role of miR-183-5p-Exo. BMSC-derived exosomal miR-183-5p could target FOXO1 to reduce apoptosis and oxidative stress in I/R cardiomyocytes and improve cardiac function, thereby protecting against MI/R injury.

Mao S ，Zhao J ，Zhang ZJ ，Zhao Q ... -  《-》