Exosomes are nanosized lipid vesicles originating from the endosomal system that carry many macromolecules from their parental cells and play important roles in intercellular communication. The functions and underlying mechanisms of exosomes in atherosclerosis have recently been intensively studied. In this review, we briefly introduce exosome biology and then focus on advances in the roles of exosomes in atherosclerosis, specifically exosomal changes associated with atherosclerosis, their cellular origins and potential functional cargos, and their detailed impacts on recipient cells. We also discuss the potential of exosomes as biomarkers and drug carriers for managing atherosclerosis.

Exosomes in atherosclerosis: performers, bystanders, biomarkers, and therapeutic targets.

Circular RNA CircHIPK3 Promotes Homeostasis of the Intestinal Epithelium by Reducing MicroRNA 29b Function.

Myocardial infarction (MI) is defined as cardiomyocyte death in a clinical context consistent with ischemic insult. MI remains one of the leading causes of morbidity and mortality worldwide. Although there are a number of effective clinical methods for the diagnosis and treatment of MI, further investigation of novel biomarkers and molecular therapeutic targets is required. Circular RNAs (circRNAs), novel non-coding RNAs, have been reported to function mainly by acting as microRNA (miRNA) sponges or binding to RNA-binding proteins (RBPs). The circRNA-miRNA-mRNA (protein) regulatory pathway regulates gene expression and affects the pathological mechanisms of various diseases. Undoubtedly, a more comprehensive understanding of the relationship between MI and circRNA will lay the foundation for the development of circRNA-based diagnostic and therapeutic strategies for MI. Therefore, this review summarizes the pathophysiological process of MI and various approaches to measure circRNA levels in MI patients, tissues, and cells; highlights the significance of circRNAs in the regulation MI pathogenesis and development; and provides potential clinical insight for the diagnosis, prognosis, and treatment of MI.

Emerging roles of circRNAs in the pathological process of myocardial infarction

Exosomes are small extracellular vesicles that are secreted by almost all types of cells and exist in almost all extracellular spaces. As an important mediator of intercellular communication, exosomes encapsulate the miRNA, lncRNA, cirRNA, mRNA, cytokine, enzyme, lipid, and other components from the cytoplasm into its closed single membrane structure and transfer them to recipient units in an autocrine, paracrine, or endocrine manner. Hypoxia is a state of low oxygen tension and is involved in many pathological processes. Hypoxia influences the size, quantity, and expression of exosome cargos. Exosomes derived from hypoxic tumor cells transfer genetics, proteins, and lipids to the recipient units to exert pleiotropic effects. Different donor cells produce different cargo contents, target different recipient units and lead to different biological effects. Hypoxic exosomes derived from tumor cells uptaken by normoxic tumor cells lead to promoted proliferation, migration, and invasion; uptaken by extracellular space or liver lead to promoted metastasis; uptaken by endothelial cells lead to promoted angiogenesis; uptaken by immune cells lead to promoted macrophage polarization and changed tumor immune microenvironment. In addition to various types of tumors, hypoxic exosomes also participate in the development of diseases in the cardiovascular system, neuron system, respiratory system, hematology system, endocrine system, urinary system, reproduction system, and skeletomuscular system. Understanding the special characteristics of hypoxic exosomes provide new insight into elaborating the pathogenesis of hypoxia related disease. This review summarizes hypoxia induced cargo changes and the biological effects of hypoxic exosomes in tumors and non-malignant diseases in different systems.

/pdf/hypoxia-induced-changes-of-exosome-cargo-and-subsequent-35hxza3v.pdf

Hypoxia Induced Changes of Exosome Cargo and Subsequent Biological Effects

Extracellular vesicles (EVs), including exosomes and microvesicles, are lipid bilayer particles naturally released from the cell. While exosomes are formed as intraluminal vesicles (ILVs) of the multivesicular endosomes (MVEs) and released to extracellular space upon MVE-plasma membrane fusion, microvesicles are generated through direct outward budding of the plasma membrane. Exosomes and microvesicles have same membrane orientation, different yet overlapping sizes; their cargo contents are selectively packed and dependent on the source cell type and functional state. Both exosomes and microvesicles can transfer bioactive RNAs, proteins, lipids, and metabolites from donor to recipient cells and influence the biological properties of the latter. Over the last decade, their potential roles as effective inter-tissue communicators in cardiovascular physiology and pathology have been increasingly appreciated. In addition, EVs are attractive sources of biomarkers for the diagnosis and prognosis of diseases, because they acquire their complex cargoes through cellular processes intimately linked to disease pathogenesis. Furthermore, EVs obtained from various stem/progenitor cell populations have been tested as cell-free therapy in various preclinical models of cardiovascular diseases and demonstrate unequivocally encouraging benefits. Here we summarize the findings from recent research on the biological functions of EVs in the ischemic heart disease and heart failure, and their potential as novel diagnostic biomarkers and therapeutic opportunities. 

Extracellular vesicles in cardiovascular disease: Biological functions and therapeutic implications

Objective: This study aimed to explore the role of circular RNAs (circRNAs) in M2 macrophage (M2M)-derived small extracellular vesicles (SEVs) in myocardial fibrosis development. Methods: The regulatory role of M2M-derived extracellular vesicles (EVs) was evaluated in a mouse model of acute myocardial infarction. Immunofluorescence, quantitative real-time PCR (RT-qPCR), nanoparticle tracking analysis, Western blot analysis and electron microscopy were used to identify macrophages, large extracellular vesicles (LEVs) and SEVs. The circRNA expression profiles of M0 macrophages (M0Ms) and M2Ms were determined by microarray analysis. Bioinformatic analysis, cell coculture and cell proliferation assays were performed to investigate the expression, function, and regulatory mechanisms of circUbe3a in vitro. qPCR, RNA immunoprecipitation (RIP), dual-luciferase reporter assays, RNA fluorescence in situ hybridization (RNA-FISH), Western blot analysis and a series of rescue experiments were used to verify the correlation among circUbe3a, miR-138-5p and RhoC. Results: CircUbe3a from M2M-derived SEVs triggered functional changes in cardiac fibroblasts (CFs). CircUbe3a was synthesized and loaded into SEVs during increased M2M infiltration after myocardial infarction. The fusion of the released SEVs with the plasma membrane likely caused the release of circUbe3a into the cytosol of CFs. Silencing or overexpressing circUbe3a altered CF proliferation, migration, and phenotypic transformation in vitro. We confirmed that circUbe3a plays a crucial role in enhancing functional changes in CFs by sponging miR-138-5p and then translationally repressing RhoC in vitro. In vivo, the addition of M2M-derived SEVs or overexpression of circUbe3a significantly exacerbated myocardial fibrosis after acute myocardial infarction, and these effects were partially abolished by circUbe3a-specific shRNA. Conclusions: Our findings suggest that M2M-derived circUbe3a-containing SEVs promote the proliferation, migration, and phenotypic transformation of CFs by directly targeting the miR-138-5p/RhoC axis, which may also exacerbate myocardial fibrosis after acute myocardial infarction.

CircUbe3a from M2 macrophage-derived small extracellular vesicles mediates myocardial fibrosis after acute myocardial infarction.

Exosomes play critical roles in mediating cell-to-cell communication by delivering noncoding RNAs (including miRNAs, lncRNAs, and circRNAs). Our previous study found that cardiomyocytes (CMs) subjected to hypoxia released circHIPK3-rich exosomes to regulate oxidative stress damage in cardiac endothelial cells. However, the role of exosomes in regulating angiogenesis after myocardial infarction (MI) remains unknown. The aim of this study was to establish the effects of exosomes derived from hypoxia-induced CMs on the migration and angiogenic tube formation of cardiac endothelial cells. Here, we reported that hypoxic exosomes (HPC-exos) can effectively reduce the infarct area and promote angiogenesis in the border surrounding the infarcted area. HPC-exos can also promote cardiac endothelial cell migration, proliferation, and tube formation in vitro. However, these effects were weakened after silencing circHIPK3 in hypoxia-induced CMs. We further verified that silencing and overexpressing circHIPK3 changed cardiac endothelial cell proliferation, migration, and tube formation in vitro by regulating the miR-29a expression. In addition, exosomal circHIPK3 derived from hypoxia-induced CMs first led to increased VEGFA expression by inhibiting miR-29a activity and then promoted accelerated cell cycle progression and proliferation in cardiac endothelial cells. Overexpression of miR-29a mimicked the effect of silencing circHIPK3 on cardiac endothelial cell activity in vitro. Thus, our study provides a novel mechanism by which exosomal circRNAs are involved in the communication between CMs and cardiac endothelial cells.

/pdf/exosomal-circhipk3-released-from-hypoxia-induced-24z1g6s7js.pdf

Exosomal CircHIPK3 Released from Hypoxia-Induced Cardiomyocytes Regulates Cardiac Angiogenesis after Myocardial Infarction.

Autophagy and apoptosis are involved in myocardial ischemia/reperfusion (I/R) injury. Research indicates that circular RNA HIPK3 (circHIPK3) is crucial to cell autophagy and apoptosis in various cancer types. However, the role of circHIPK3 in the regulation of cardiomyocyte autophagy and apoptosis during I/R remains unknown. Our study aimed to examine the regulatory effect of circHIPK3 during myocardial I/R and investigate its mechanism in cardiomyocyte autophagy and apoptosis. Methods and results. The expression of circHIPK3 was upregulated during myocardial I/R injury and hypoxia/reoxygenation (H/R) injury of cardiomyocytes. To study the potential role of circHIPK3 in myocardial H/R injury, we performed gain-of-function and loss-of-function analyses of circHIPK3 in cardiomyocytes. Overexpression of circHIPK3 significantly promoted H/R-induced cardiomyocyte autophagy and cell injury (increased intracellular reactive oxygen species (ROS) and apoptosis) compared to those in the control group, while silencing of circHIPK3 showed the opposite effect. Further research found that circHIPK3 acted as an endogenous miR-20b-5p sponge to sequester and inhibit miR-20b-5p activity, resulting in increased ATG7 expression. In addition, miR-20b-5p inhibitors reversed the decrease in ATG7 induced by silencing circHIPK3. Conclusions. CircHIPK3 can accelerate cardiomyocyte autophagy and apoptosis during myocardial I/R injury through the miR-20b-5p/ATG7 axis. These data suggest that circHIPK3 may serve as a potential therapeutic target for I/R.

/pdf/circhipk3-regulates-the-autophagy-and-apoptosis-of-hypoxia-1lngn9777d.pdf

CircHIPK3 regulates the autophagy and apoptosis of hypoxia/reoxygenation-stimulated cardiomyocytes via the miR-20b-5p/ATG7 axis.

To investigate the mechanism by which hypoxia-reoxygenation (HR) mediates macrophage polarization to the M1 phenotype and then mediates cardiomyocyte (CM) pyroptosis through exosome release. Mouse bone marrow macrophages and CMs were cultured in vitro under hypoxia for 12 h and reoxygenation for 6 h to establish an HR cell model. qPCR was used to detect the M1 or M2 macrophage markers IL-1β, TNF-α, MR, and Arg, and a macrophage and CM coculture system was then established. Macrophages were transfected with an exosome-CD63-red fluorescent protein (RFP) lentivirus, allowing secretion of exosomes expressing RFP, and GW4869 was used to inhibit exosome release by macrophages. qPCR detected miR-29 expression in macrophage-derived exosomes, and macrophages were transfected with miR-29a inhibitors to obtain exosomes with low miR-29a expression (siR-exos). Pyroptosis indicators were detected by Western blot and ELISA. Importantly, LPS induced bone marrow macrophage polarization to the M1 type as a positive control to further verify that these exosomes (LPS-exos) regulated CM pyroptosis by delivering miR29a. Dual luciferase reporter and Western blot assays were adopted to analyze the miR-29a and MCL-1 target relationship. In addition, MCL-1 overexpression was used as a rescue experiment to determine whether miR-29a regulates pyroptosis in CM by targeting MCL-1. Macrophages expressed the M1 macrophage markers IL-1β and TNF-α after HR exposure. After CM coculture, RFP expression was significantly higher in the HR group than in the normal (Nor) group but significantly reduced in the GW4869 group. Immunofluorescence showed that caspase-1 mRNA and protein expression in the HR group was significantly higher than that in the Nor group (P < 0.05). Caspase-1 expression was significantly decreased in the GW4869 group compared with the HR group (P < 0.05). Western blotting showed that the pyrolysis-related NLRP3 and ASC protein expression levels were significantly upregulated in the HR group compared with the control (Ctr) and Nor groups (P < 0.05). However, GW4869 effectively inhibited pyroptosis-related protein expression (P < 0.05). In addition, ELISA showed that the expression of the inflammation indicators IL-1β and IL-18 was significantly increased in the HR group compared to the Ctr group (P < 0.05) but decreased in the GW4869 group (P < 0.05). qPCR showed that miR-29a was upregulated in the HR group compared to the Nor group. Moreover, HR-induced exosomes (HR-exos) from macrophages exacerbated HR-induced CM pyroptosis, while inhibition of miR-29a in exosomes partially offset CM pyroptosis induction. LPS-exos promoted pyroptosis-related protein expression, as the IL-1β and IL-18 concentrations were increased in the LPS-exos group. However, pyroptosis-related proteins were observably decreased, and IL-1β and IL-18 were also significantly decreased after miR-29a inhibition when compared with that in the HR-exos and LPS-exos groups. Mcl-1 overexpression reversed miR-29a-mediated CM pyroptosis in an HR environment. HR treatment induced macrophage polarization towards the M1 phenotype, which mediated CM pyroptosis through exosomal miR-29a transfer by targeting MCL-1.

Chaofu Li

Papers

CircUbe3a from M2 macrophage-derived small extracellular vesicles mediates myocardial fibrosis after acute myocardial infarction.

Exosomal CircHIPK3 Released from Hypoxia-Induced Cardiomyocytes Regulates Cardiac Angiogenesis after Myocardial Infarction.

CircHIPK3 regulates the autophagy and apoptosis of hypoxia/reoxygenation-stimulated cardiomyocytes via the miR-20b-5p/ATG7 axis.

Hypoxia-reoxygenation induces macrophage polarization and causes the release of exosomal miR-29a to mediate cardiomyocyte pyroptosis