Mesenchymal stem cells (MSCs) have been extensively investigated for the treatment of various diseases. The therapeutic potential of MSCs is attributed to complex cellular and molecular mechanisms of action including differentiation into multiple cell lineages and regulation of immune responses via immunomodulation. The plasticity of MSCs in immunomodulation allow these cells to exert different immune effects depending on different diseases. Understanding the biology of MSCs and their role in treatment is critical to determine their potential for various therapeutic applications and for the development of MSC-based regenerative medicine. This review summarizes the recent progress of particular mechanisms underlying the tissue regenerative properties and immunomodulatory effects of MSCs. We focused on discussing the functional roles of paracrine activities, direct cell–cell contact, mitochondrial transfer, and extracellular vesicles related to MSC-mediated effects on immune cell responses, cell survival, and regeneration. This will provide an overview of the current research on the rapid development of MSC-based therapies.

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Mechanisms underlying the protective effects of mesenchymal stem cell-based therapy.

Mesenchymal stem cell (MSC)-sourced secretome, defined as the set of MSC-derived bioactive factors (soluble proteins, nucleic acids, lipids and extracellular vesicles), showed therapeutic effects similar to those observed after transplantation of MSCs. MSC-derived secretome may bypass many side effects of MSC-based therapy, including unwanted differentiation of engrafted MSCs. In contrast to MSCs which had to be expanded in culture to reach optimal cell number for transplantation, MSC-sourced secretome is immediately available for treatment of acute conditions, including fulminant hepatitis, cerebral ischemia and myocardial infarction. Additionally, MSC-derived secretome could be massively produced from commercially available cell lines avoiding invasive cell collection procedure. In this review article we emphasized molecular and cellular mechanisms that were responsible for beneficial effects of MSC-derived secretomes in the treatment of degenerative and inflammatory diseases of hepatobiliary, respiratory, musculoskeletal, gastrointestinal, cardiovascular and nervous system. Results obtained in a large number of studies suggested that administration of MSC-derived secretomes represents a new, cell-free therapeutic approach for attenuation of inflammatory and degenerative diseases. Therapeutic effects of MSC-sourced secretomes relied on their capacity to deliver genetic material, growth and immunomodulatory factors to the target cells enabling activation of anti-apoptotic and pro-survival pathways that resulted in tissue repair and regeneration.

Molecular Mechanisms Responsible for Therapeutic Potential of Mesenchymal Stem Cell-Derived Secretome

Extracellular vesicles (EVs) are a heterogeneous group of natural particles that are relevant to the treatment of cardiovascular diseases. These endogenous vesicles have certain properties that allow them to survive in the extracellular space, bypass biological barriers and deliver their biologically active molecular cargo to recipient cells. Moreover, EVs can be bioengineered to increase their stability, bioactivity, presentation to acceptor cells and capacity for on-target binding at both cell-type-specific and tissue-specific levels. Bioengineering of EVs involves the modification of the donor cell before EV isolation or direct modification of the EV properties after isolation. The therapeutic potential of native EVs and bioengineered EVs has been only minimally explored in the context of cardiovascular diseases. Efforts to harness the therapeutic potential of EVs will require innovative approaches and a comprehensive integration of knowledge gathered from decades of research into molecular-compound delivery. In this Review, we outline the endogenous properties of EVs that make them natural delivery agents as well as the features that can be improved by bioengineering. We also discuss the therapeutic applications of native and bioengineered EVs to cardiovascular diseases and examine the opportunities and challenges that need to be addressed to advance this research area, with an emphasis on clinical translation. Extracellular vesicles are a heterogeneous group of natural particles that can deliver their biologically active molecular cargo to recipient cells. In this Review, the authors outline the endogenous properties of extracellular vesicles that make them natural delivery agents and the features that can be improved by bioengineering for the treatment of cardiovascular diseases.

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Native and bioengineered extracellular vesicles for cardiovascular therapeutics

The therapeutic benefits of mesenchymal stromal cell (MSC) transplantation have been attributed to their secreted factors, including extracellular vesicles (EVs) and soluble factors. The potential of employing the MSC secretome as an alternative acellular approach to cell therapy is being investigated in various tissue injury indications, but EVs administered via bolus injections are rapidly sequestered and cleared. However, biomaterials offer delivery platforms to enhance EV retention rates and healing efficacy. In this review, we highlight the mechanisms underpinning the therapeutic effects of MSC-EVs and soluble factors as effectors of immunomodulation and tissue regeneration, conferred primarily via their nucleic acid and protein contents. We discuss how manipulating the cell culture microenvironment or genetic modification of MSCs can further augment the potency of their secretions. The most recent advances in the development of EV-functionalized biomaterials that mediate enhanced angiogenesis and cell survival, while attenuating inflammation and fibrosis, are presented. Finally, some technical challenges to be considered for the clinical translation of biomaterials carrying MSC-secreted bioactive cargo are discussed.

https://onlinelibrary.wiley.com/doi/epdf/10.1002/adfm.201909125

Biomaterials functionalized with MSC secreted extracellular vesicles and soluble factors for tissue regeneration

Stem cell-derived exosomes have been recognized as a potential therapy for cardiovascular disease. However, the low retention rate of exosomes after transplantation in vivo remains a major challenge in clinical applications. The aim of this study is to investigate whether human umbilical cord mesenchymal stem cell derived exosomes (UMSC-Exo) encapsulated in functional peptide hydrogels could increase the retention and stability of exosomes and improve heart function in a rat myocardial infarction model. Our results demonstrated that the PA-GHRPS peptide protected H9C2 cells from H2O2-induced oxidative stress. The gelatinization ability of PA-GHRPS can be enhanced by peptide NapFF. Therefore, these two peptides were mixed to form the PGN hydrogel, which was used to encapsulate exosomes. Our data showed that the PGN hydrogel was able to encapsulate exosomes effectively and ensured a stable and sustained release of exosomes. The exosome/PGN hydrogel mixture was injected into the infarcted border zone of rat hearts. Compared to the exosome treatment alone, the mixture improved the myocardial function by reducing inflammation, fibrosis and apoptosis, and by promoting angiogenesis. The strategy used in this study provided a practical and effective method to harness exosomes for myocardial regeneration.

Human umbilical cord mesenchymal stem cell derived exosomes encapsulated in functional peptide hydrogels promote cardiac repair.

Exosomes (Exo) secreted from hypoxia-conditioned bone marrow mesenchymal stem cells (BM-MSCs) were found to be protective for ischemic disease. However, the role of exosomal miRNA in the protective effect of hypoxia-conditioned BM-MSCs-derived Exo (Hypo-Exo) remains largely uncharacterized and the poor specificity of tissue targeting of Exo limits their clinical applications. Therefore, the objective of this study was to examine the effect of miRNA in Hypo-Exo on the repair of ischemic myocardium and its underlying mechanisms. We further developed modified Hypo-Exo with high specificity to the myocardium and evaluate its therapeutic effects. Methods: Murine BM-MSCs were subjected to hypoxia or normoxia culture and Exo were subsequently collected. Hypo-Exo or normoxia-conditioned BM-MSC-derived Exo (Nor-Exo) were administered to mice with permanent condition of myocardial infarction (MI). After 28 days, to evaluate the therapeutic effects of Hypo-Exo, infarction area and cardio output in Hypo-Exo and Nor-Exo treated MI mice were compared through Masson's trichrome staining and echocardiography respectively. We utilized the miRNA array to identify the significantly differentially expressed miRNAs between Nor-Exo and Hypo-Exo. One of the most enriched miRNA in Hypo-Exo was knockdown by applying antimiR in Hypoxia-conditioned BM-MSCs. Then we performed intramyocardial injection of candidate miRNA-knockdown-Hypo-Exo in a murine MI model, changes in the candidate miRNA's targets expression of cardiomyocytes and the cardiac function were characterized. We conjugated Hypo-Exo with an ischemic myocardium-targeted (IMT) peptide by bio-orthogonal chemistry, and tested its targeting specificity and therapeutic efficiency via systemic administration in the MI mice. Results: The miRNA array revealed significant enrichment of miR-125b-5p in Hypo-Exo compared with Nor-Exo. Administration of miR-125b knockdown Hypo-Exo significantly increased the infarction area and suppressed cardiomyocyte survival post-MI. Mechanistically, miR-125b knockdown Hypo-Exo lost the capability to suppress the expression of the proapoptotic genes p53 and BAK1 in cardiomyocytes. Intravenous administration of IMT-conjugated Hypo-Exo (IMT-Exo) showed specific targeting to the ischemic lesions in the injured heart and exerted a marked cardioprotective function post-MI. Conclusion: Our results illustrate a new mechanism by which Hypo-Exo-derived miR125b-5p facilitates ischemic cardiac repair by ameliorating cardiomyocyte apoptosis. Furthermore, our IMT- Exo may serve as a novel drug carrier that enhances the specificity of drug delivery for ischemic disease.

Hypoxia-elicited mesenchymal stem cell-derived exosomes facilitates cardiac repair through miR-125b-mediated prevention of cell death in myocardial infarction

Angiogenesis is critical for re-establishing the blood supply to the surviving myocardium after myocardial infarction (MI) in patients with acute coronary syndrome (ACS). MicroRNAs are recognised as important epigenetic regulators of endothelial function. The aim of this study was to determine the roles of microRNAs in angiogenesis. Eighteen circulating microRNAs including miR-185-5p were differently expressed in plasma from patients with ACS by high-throughput RNA sequencing. The expressional levels of miR-185-5p were dramatically reduced in hearts isolated from mice following MI and cultured human umbilical vein endothelial cells (HUVECs) under hypoxia, as determined by fluorescence in situ hybridisation and quantitative RT-PCR. Evidence from computational prediction and luciferase reporter gene activity indicated that cathepsin K (CatK) mRNA is a target of miR-185-5p. In HUVECs, miR-185-5p mimics inhibited cell proliferations, migrations and tube formations under hypoxia, while miR-185-5p inhibitors performed the opposites. Further, the inhibitory effects of miR-185-5p up-regulation on cellular functions of HUVECs were abolished by CatK gene overexpression, and adenovirus-mediated CatK gene silencing ablated these enhancive effects in HUVECs under hypoxia. In vivo studies indicated that gain-function of miR-185-5p by agomir infusion down-regulated CatK gene expression, impaired angiogenesis and delayed the recovery of cardiac functions in mice following MI. These actions of miR-185-5p agonists were mirrored by in vivo knockdown of CatK in mice with MI. Endogenous reductions of miR-185-5p in endothelial cells induced by hypoxia increase CatK gene expression to promote angiogenesis and to accelerate the recovery of cardiac function in mice following MI.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/jcmm.14016

Endogenous reduction of miR-185 accelerates cardiac function recovery in mice following myocardial infarction via targeting of cathepsin K.

Aim The slow coronary flow (SCF) phenomenon was characterized by delayed perfusion of epicardial arteries, and no obvious coronary artery lesion in coronary angiography. The prognosis of patients with slow coronary flow was poor. However, there is lack of rapid, simple, and accurate method for SCF diagnosis. This study aimed to explore the utility of plasma choline as a diagnostic biomarker for SCF. Methods Patients with coronary artery stenosis <40% evaluated by the coronary angiogram method were recruited in this study and were grouped into normal coronary flow (NCF) and SCF by thrombolysis in myocardial infarction frame count (TFC). Plasma choline concentrations of patients with NCF and SCF were quantified by Ultra Performance Liquid Chromatography Tandem Mass Spectrometry. Correlation analysis was performed between plasma choline concentration and TFC. Receiver operating characteristic (ROC) curve analysis with or without confounding factor adjustment was applied to predict the diagnostic power of plasma choline in SCF. Results Forty-four patients with SCF and 21 patients with NCF were included in this study. TFC in LAD, LCX, and RCA and mean TFC were significantly higher in patients with SCF in comparison with patients with NCF (32.67 ± 8.37 vs. 20.66 ± 3.41, P  <  0.01). Plasma choline level was obviously higher in patients with SCF when compared with patients with NCF (754.65 ± 238.18 vs. 635.79 ± 108.25, P=0.007). Plasma choline level had significantly positive correlation with Mean TFC (r = 0.364, P=0.002). Receiver operating characteristic (ROC) analysis showed that choline with or without confounding factor adjustment had an AUC score of 0.65 and 0.77, respectively. Conclusions TFC were closely related with plasma choline level, and plasma choline can be a suitable and stable diagnostic biomarker for SCF.

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Plasma Choline as a Diagnostic Biomarker in Slow Coronary Flow

Background Prophylactic exercise improves clinical outcomes in patients experiencing severe ischemic diseases. Previous studies have shown that exercise could alter the amount or content of circulating exosomes. However, little is known about the role of precursory exercise-derived circulating exosomes (Exe-Exo) in ischemic diseases. We therefore aimed to explore the function and mechanism of Exe-Exo in endogenous revascularization and perfusion recovery in peripheral arterial disease. Methods and Results We first determined that 4 weeks of precursory treadmill exercise improved perfusion recovery on days 7, 14 and 21 after unilateral femoral artery ligation (FAL) but had no effect immediately after ligation. Then, local muscle delivery of Exe-Exo promotes arteriogenesis, angiogenesis and perfusion recovery, which could be abolished by GW4869, a well-recognized pharmacological agent inhibiting exosome release. This suggests that Exe-Exo mediated exercise-induced revascularization. In vitro, Exe-Exo enhanced endothelial cell proliferation, migration and tube formation. In addition, we identified miR-125a-5p as a novel exerkine through exosomal miRNA sequencing and RT-qPCR validation. Inhibition of miR-125a-5p abrogated the beneficial effects of Exe-Exo both in vivo and in vitro. Mechanistically, these exercise-afforded benefits were attributed to the exosomal miR-125a-5p downregulation of ECE1 expression and the subsequent activation of the AKT/eNOS downstream signaling pathway. Specifically, skeletal muscle may be a major tissue source of exercise-induced exosomal miR-125a-5p via fluorescence in situ hybridization. Conclusions Endogenous circulating exosomal miR-125a-5p promotes exercise-induced revascularization via targeting ECE1 and activating AKT/eNOS downstream signaling pathway. Identify exosomal miR-125a-5p as a novel exerkine, and highlight its potential therapeutic role in the prevention and treatment of peripheral arterial disease. Graphical Abstract Proposed mechanisms by which exercise-induced circulating exosomes promote arteriogenesis and angiogenesis after lower limb ischemia. Four weeks of exercise training mainly increased miR-125a-5p expression in adductor muscles and cargoes into exercise-derived exosomes. These miR-125a-5p-enriched exosomes were delivered to endothelial cells and enhanced blood perfusion recovery after femoral artery ligation by targeting ECE1 and activating the AKT/eNOS signaling pathway.

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Prophylactic exercise-derived circulating exosomal miR-125a-5p promotes endogenous revascularization after hindlimb ischemia by targeting endothelin converting enzyme 1

Xue-Ting Qiu

Papers

Hypoxia-elicited mesenchymal stem cell-derived exosomes facilitates cardiac repair through miR-125b-mediated prevention of cell death in myocardial infarction

Endogenous reduction of miR-185 accelerates cardiac function recovery in mice following myocardial infarction via targeting of cathepsin K.

Plasma Choline as a Diagnostic Biomarker in Slow Coronary Flow

Prophylactic exercise-derived circulating exosomal miR-125a-5p promotes endogenous revascularization after hindlimb ischemia by targeting endothelin converting enzyme 1

Omentin-1 drives cardiomyocyte cell cycle arrest and metabolic maturation by interacting with BMP7