Extracellular Vesicle-Encapsulated Adeno-Associated Viruses for Therapeutic Gene Delivery to the Heart.

BACKGROUND: Adeno-associated virus (AAV) has emerged as one of the best tools for cardiac gene delivery due to its cardiotropism, long-term expression, and safety. However, a significant challenge to its successful clinical use is preexisting neutralizing antibodies (NAbs), which bind to free AAVs, prevent efficient gene transduction, and reduce or negate therapeutic effects. Here we describe extracellular vesicle-encapsulated AAVs (EV-AAVs), secreted naturally by AAV-producing cells, as a superior cardiac gene delivery vector that delivers more genes and offers higher NAb resistance. METHODS: We developed a 2-step density-gradient ultracentrifugation method to isolate highly purified EV-AAVs. We compared the gene delivery and therapeutic efficacy of EV-AAVs with an equal titer of free AAVs in the presence of NAbs, both in vitro and in vivo. In addition, we investigated the mechanism of EV-AAV uptake in human left ventricular and human induced pluripotent stem cell-derived cardiomyocytes in vitro and mouse models in vivo using a combination of biochemical techniques, flow cytometry, and immunofluorescence imaging. RESULTS: Using cardiotropic AAV serotypes 6 and 9 and several reporter constructs, we demonstrated that EV-AAVs deliver significantly higher quantities of genes than AAVs in the presence of NAbs, both to human left ventricular and human induced pluripotent stem cell-derived cardiomyocytes in vitro and to mouse hearts in vivo. Intramyocardial delivery of EV-AAV9-sarcoplasmic reticulum calcium ATPase 2a to infarcted hearts in preimmunized mice significantly improved ejection fraction and fractional shortening compared with AAV9-sarcoplasmic reticulum calcium ATPase 2a delivery. These data validated NAb evasion by and therapeutic efficacy of EV-AAV9 vectors. Trafficking studies using human induced pluripotent stem cell-derived cells in vitro and mouse hearts in vivo showed significantly higher expression of EV-AAV6/9-delivered genes in cardiomyocytes compared with noncardiomyocytes, even with comparable cellular uptake. Using cellular subfraction analyses and pH-sensitive dyes, we discovered that EV-AAVs were internalized into acidic endosomal compartments of cardiomyocytes for releasing and acidifying AAVs for their nuclear uptake. CONCLUSIONS: Together, using 5 different in vitro and in vivo model systems, we demonstrate significantly higher potency and therapeutic efficacy of EV-AAV vectors compared with free AAVs in the presence of NAbs. These results establish the potential of EV-AAV vectors as a gene delivery tool to treat heart failure.

Therapeutic and Diagnostic Translation of Extracellular Vesicles in Cardiovascular Diseases: Roadmap to the Clinic.

Exosomes are small membrane-bound vesicles of endocytic origin that are actively secreted. The potential of exosomes as effective communicators of biological signaling in myocardial function has previously been investigated, and a recent explosion in exosome research not only underscores their significance in cardiac physiology and pathology, but also draws attention to methodological limitations of studying these extracellular vesicles. In this review, we discuss recent advances and challenges in exosome research with an emphasis on scientific innovations in isolation, identification, and characterization methodologies, and we provide a comprehensive summary of web-based resources available in the field. Importantly, we focus on the biology and function of exosomes, highlighting their fundamental role in cardiovascular pathophysiology to further support potential applications of exosomes as biomarkers and therapeutics for cardiovascular diseases.

Regulation of the Methylation and Expression Levels of the BMPR2 Gene by SIN3a as a Novel Therapeutic Mechanism in Pulmonary Arterial Hypertension.

BACKGROUND: Epigenetic mechanisms are critical in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have suggested that hypermethylation of the BMPR2 (bone morphogenetic protein receptor type 2) promoter is associated with BMPR2 downregulation and progression of PAH. Here, we investigated for the first time the role of SIN3a (switch-independent 3a), a transcriptional regulator, in the epigenetic mechanisms underlying hypermethylation of BMPR2 in the pathogenesis of PAH. METHODS: We used lung samples from PAH patients and non-PAH controls, preclinical mouse and rat PAH models, and human pulmonary arterial smooth muscle cells. Expression of SIN3a was modulated using a lentiviral vector or a siRNA in vitro and a specific adeno-associated virus serotype 1 or a lentivirus encoding for human SIN3a in vivo. RESULTS: SIN3a is a known transcriptional regulator; however, its role in cardiovascular diseases, especially PAH, is unknown. It is interesting that we detected a dysregulation of SIN3 expression in patients and in rodent models, which is strongly associated with decreased BMPR2 expression. SIN3a is known to regulate epigenetic changes. Therefore, we tested its role in the regulation of BMPR2 and found that BMPR2 is regulated by SIN3a. It is interesting that SIN3a overexpression inhibited human pulmonary arterial smooth muscle cells proliferation and upregulated BMPR2 expression by preventing the methylation of the BMPR2 promoter region. RNA-sequencing analysis suggested that SIN3a downregulated the expression of DNA and histone methyltransferases such as DNMT1 (DNA methyltransferase 1) and EZH2 (enhancer of zeste 2 polycomb repressive complex 2) while promoting the expression of the DNA demethylase TET1 (ten-eleven translocation methylcytosine dioxygenase 1). Mechanistically, SIN3a promoted BMPR2 expression by decreasing CTCF (CCCTC-binding factor) binding to the BMPR2 promoter. Last, we identified intratracheal delivery of adeno-associated virus serotype human SIN3a to be a beneficial therapeutic approach in PAH by attenuating pulmonary vascular and right ventricle remodeling, decreasing right ventricle systolic pressure and mean pulmonary arterial pressure, and restoring BMPR2 expression in rodent models of PAH. CONCLUSIONS: All together, our study unveiled the protective and beneficial role of SIN3a in pulmonary hypertension. We also identified a novel and distinct molecular mechanism by which SIN3a regulates BMPR2 in human pulmonary arterial smooth muscle cells. Our study also identified lung-targeted SIN3a gene therapy using adeno-associated virus serotype 1 as a new promising therapeutic strategy for treating patients with PAH.

Bioinspired artificial exosomes based on lipid nanoparticles carrying let-7b-5p promote angiogenesis in vitro and in vivo.

MicroRNAs (miRNAs) regulate gene expression by post-transcriptional inhibition of target genes. Proangiogenic small extracellular vesicles (sEVs; popularly identified with the name "exosomes") with a composite cargo of miRNAs are secreted by cultured stem cells and present in human biological fluids. Lipid nanoparticles (LNPs) represent an advanced platform for clinically approved delivery of RNA therapeutics. In this study, we aimed to (1) identify the miRNAs responsible for sEV-induced angiogenesis; (2) develop the prototype of bioinspired "artificial exosomes" (AEs) combining LNPs with a proangiogenic miRNA, and (3) validate the angiogenic potential of the bioinspired AEs. We previously reported that human sEVs from bone marrow (BM)-CD34+ cells and pericardial fluid (PF) are proangiogenic. Here, we have shown that sEVs secreted from saphenous vein pericytes and BM mesenchymal stem cells also promote angiogenesis. Analysis of miRNA datasets available in-house or datamined from GEO identified the let-7 family as common miRNA signature of the proangiogenic sEVs. LNPs with either hsa-let-7b-5p or cyanine 5 (Cy5)-conjugated Caenorhabditis elegans miR-39 (Cy5-cel-miR-39; control miRNA) were prepared using microfluidic micromixing. let-7b-5p-AEs did not cause toxicity and transferred functionally active let-7b-5p to recipient endothelial cells (ECs). let-7b-AEs also improved EC survival under hypoxia and angiogenesis in vitro and in vivo. Bioinspired proangiogenic AEs could be further developed into innovative nanomedicine products targeting ischemic diseases.

Analysis of extracellular vesicle miRNA profiles in heart failure.

Extracellular vesicles (EVs) have recently emerged as an important carrier for various genetic materials including microRNAs (miRs). Growing evidences suggested that several miRs transported by EVs were particularly involved in modulating cardiac function. However, it has remained unclear what miRs are enriched in EVs and play an important role in the pathological condition. Therefore, we established the miR expression profiles in EVs from murine normal and failing hearts and consecutively identified substantially altered miRs. In addition, we have performed bioinformatics approach to predict potential cardiac outcomes through the identification of miR targets. Conclusively, we observed approximately 63% of predicted targets were validated with previous reports. Notably, the predicted targets by this approach were often involved in both beneficial and malicious signalling pathways, which may reflect heterogeneous cellular origins of EVs in tissues. Lastly, there has been an active debate on U6 whether it is a proper control. Through further analysis of EV miR profiles, miR-676 was identified as a superior reference control due to its consistent and abundant expressions. In summary, our results contribute to identifying specific EV miRs for the potential therapeutic targets in heart failure and suggest that miR-676 as a new reference control for the EV miR studies.

FTO-Dependent N6-Methyladenosine Regulates Cardiac Function During Remodeling and Repair.

BACKGROUND: Despite its functional importance in various fundamental bioprocesses, studies of N6-methyladenosine (m6A) in the heart are lacking. Here, we show that the FTO (fat mass and obesity-associated protein), an m6A demethylase, plays a critical role in cardiac contractile function during homeostasis, remodeling, and regeneration. METHODS: We used clinical human samples, preclinical pig and mouse models, and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO by using adeno-associated virus serotype 9 (in vivo), adenovirus (both in vivo and in vitro), and small interfering RNAs (in vitro) to study its function in regulating cardiomyocyte m6A, calcium dynamics and contractility, and cardiac function postischemia. We performed methylated (m6A) RNA immunoprecipitation sequencing to map transcriptome-wide m6A, and methylated (m6A) RNA immunoprecipitation quantitative polymerase chain reaction assays to map and validate m6A in individual transcripts, in healthy and failing hearts, and in myocytes. RESULTS: We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is performed by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. In addition, we demonstrate that FTO overexpression in mouse models of myocardial infarction decreased fibrosis and enhanced angiogenesis. CONCLUSIONS: Collectively, our study demonstrates the functional importance of the FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.

Physiologic, Pathologic, and Therapeutic Paracrine Modulation of Cardiac Excitation-Contraction Coupling.

Cardiac excitation-contraction coupling (ECC) is the orchestrated process of initial myocyte electrical excitation, which leads to calcium entry, intracellular trafficking, and subsequent sarcomere shortening and myofibrillar contraction. Neurohumoral ß-adrenergic signaling is a well-established mediator of ECC; other signaling mechanisms, such as paracrine signaling, have also demonstrated significant impact on ECC but are less well understood. For example, resident heart endothelial cells are well-known physiological paracrine modulators of cardiac myocyte ECC mainly via NO and endothelin-1. Moreover, recent studies have demonstrated other resident noncardiomyocyte heart cells (eg, physiological fibroblasts and pathological myofibroblasts), and even experimental cardiotherapeutic cells (eg, mesenchymal stem cells) are also capable of altering cardiomyocyte ECC through paracrine mechanisms. In this review, we first focus on the paracrine-mediated effects of resident and therapeutic noncardiomyocytes on cardiomyocyte hypertrophy, electrophysiology, and calcium handling, each of which can modulate ECC, and then discuss the current knowledge about key paracrine factors and their underlying mechanisms of action. Next, we provide a case example demonstrating the promise of tissue-engineering approaches to study paracrine effects on tissue-level contractility. More specifically, we present new functional and molecular data on the effects of human adult cardiac fibroblast conditioned media on human engineered cardiac tissue contractility and ion channel gene expression that generally agrees with previous murine studies but also suggests possible species-specific differences. By contrast, paracrine secretions by human dermal fibroblasts had no discernible effect on human engineered cardiac tissue contractile function and gene expression. Finally, we discuss systems biology approaches to help identify key stem cell paracrine mediators of ECC and their associated mechanistic pathways. Such integration of tissue-engineering and systems biology methods shows promise to reveal novel insights into paracrine mediators of ECC and their underlying mechanisms of action, ultimately leading to improved cell-based therapies for patients with heart disease.

miR-146a Suppresses SUMO1 Expression and Induces Cardiac Dysfunction in Maladaptive Hypertrophy.

RATIONALE: Abnormal SUMOylation has emerged as a characteristic of heart failure (HF) pathology. Previously, we found reduced SUMO1 (small ubiquitin-like modifier 1) expression and SERCA2a (sarcoplasmic reticulum Ca2+-ATPase) SUMOylation in human and animal HF models. SUMO1 gene delivery or small molecule activation of SUMOylation restored SERCA2a SUMOylation and cardiac function in HF models. Despite the critical role of SUMO1 in HF, the regulatory mechanisms underlying SUMO1 expression are largely unknown. OBJECTIVE: To examine miR-146a-mediated SUMO1 regulation and its consequent effects on cardiac morphology and function. METHODS AND RESULTS: In this study, miR-146a was identified as a SUMO1-targeting microRNA in the heart. A strong correlation was observed between miR-146a and SUMO1 expression in failing mouse and human hearts. miR-146a was manipulated in cardiomyocytes through AAV9 (adeno-associated virus serotype 9)-mediated gene delivery, and cardiac morphology and function were analyzed by echocardiography and hemodynamics. Overexpression of miR-146a reduced SUMO1 expression, SERCA2a SUMOylation, and cardiac contractility in vitro and in vivo. The effects of miR-146a inhibition on HF pathophysiology were examined by transducing a tough decoy of miR-146a into mice subjected to transverse aortic constriction. miR-146a inhibition improved cardiac contractile function and normalized SUMO1 expression. The regulatory mechanisms of miR-146a upregulation were elucidated by examining the major miR-146a-producing cell types and transfer mechanisms. Notably, transdifferentiation of fibroblasts triggered miR-146a overexpression and secretion through extracellular vesicles, and the extracellular vesicle-associated miR-146a transfer was identified as the causative mechanism of miR-146a upregulation in failing cardiomyocytes. Finally, extracellular vesicles isolated from failing hearts were shown to contain high levels of miR-146a and exerted negative effects on the SUMO1/SERCA2a signaling axis and hence cardiomyocyte contractility. CONCLUSIONS: Taken together, our results show that miR-146a is a novel regulator of the SUMOylation machinery in the heart, which can be targeted for therapeutic intervention.

Exosomal microRNA-21-5p Mediates Mesenchymal Stem Cell Paracrine Effects on Human Cardiac Tissue Contractility.

RATIONALE: The promising clinical benefits of delivering human mesenchymal stem cells (hMSCs) for treating heart disease warrant a better understanding of underlying mechanisms of action. hMSC exosomes increase myocardial contractility; however, the exosomal cargo responsible for these effects remains unresolved. OBJECTIVE: This study aims to identify lead cardioactive hMSC exosomal microRNAs to provide a mechanistic basis for optimizing future stem cell-based cardiotherapies. METHODS AND RESULTS: Integrating systems biology and human engineered cardiac tissue (hECT) technologies, partial least squares regression analysis of exosomal microRNA profiling data predicted microRNA-21-5p (miR-21-5p) levels positively correlate with contractile force and calcium handling gene expression responses in hECTs treated with conditioned media from multiple cell types. Furthermore, miR-21-5p levels were significantly elevated in hECTs treated with the exosome-enriched fraction of the hMSC secretome (hMSC-exo) versus untreated controls. This motivated experimentally testing the human-specific role of miR-21-5p in hMSC-exo-mediated increases of cardiac tissue contractility. Treating hECTs with miR-21-5p alone was sufficient to recapitulate effects observed with hMSC-exo on hECT developed force and expression of associated calcium handling genes (eg, SERCA2a and L-type calcium channel). Conversely, knockdown of miR-21-5p in hMSCs significantly diminished exosomal procontractile and associated calcium handling gene expression effects on hECTs. Western blots supported miR-21-5p effects on calcium handling gene expression at the protein level, corresponding to significantly increased calcium transient amplitude and decreased decay time constant in comparison to miR-scramble control. Mechanistically, cotreating with miR-21-5p and LY294002, a PI3K inhibitor, suppressed these effects. Finally, mathematical simulations predicted the translational capacity for miR-21-5p treatment to restore calcium handling in mature ischemic adult human cardiomyocytes. CONCLUSIONS: miR-21-5p plays a key role in hMSC-exo-mediated effects on cardiac contractility and calcium handling, likely via PI3K signaling. These findings may open new avenues of research to harness the role of miR-21-5p in optimizing future stem cell-based cardiotherapies.

Angiogenic Mechanisms of Human CD34+ Stem Cell Exosomes in the Repair of Ischemic Hindlimb.

RATIONALE: Paracrine secretions seem to mediate therapeutic effects of human CD34+ stem cells locally transplanted in patients with myocardial and critical limb ischemia and in animal models. Earlier, we had discovered that paracrine secretion from human CD34+ cells contains proangiogenic, membrane-bound nanovesicles called exosomes (CD34Exo). OBJECTIVE: Here, we investigated the mechanisms of CD34Exo-mediated ischemic tissue repair and therapeutic angiogenesis by studying their miRNA content and uptake. METHODS AND RESULTS: When injected into mouse ischemic hindlimb tissue, CD34Exo, but not the CD34Exo-depleted conditioned media, mimicked the beneficial activity of their parent cells by improving ischemic limb perfusion, capillary density, motor function, and their amputation. CD34Exo were found to be enriched with proangiogenic miRNAs such as miR-126-3p. Knocking down miR-126-3p from CD34Exo abolished their angiogenic activity and beneficial function both in vitro and in vivo. Interestingly, injection of CD34Exo increased miR-126-3p levels in mouse ischemic limb but did not affect the endogenous synthesis of miR-126-3p, suggesting a direct transfer of stable and functional exosomal miR-126-3p. miR-126-3p enhanced angiogenesis by suppressing the expression of its known target, SPRED1, simultaneously modulating the expression of genes involved in angiogenic pathways such as VEGF (vascular endothelial growth factor), ANG1 (angiopoietin 1), ANG2 (angiopoietin 2), MMP9 (matrix metallopeptidase 9), TSP1 (thrombospondin 1), etc. Interestingly, CD34Exo, when treated to ischemic hindlimbs, were most efficiently internalized by endothelial cells relative to smooth muscle cells and fibroblasts, demonstrating a direct role of stem cell-derived exosomes on mouse endothelium at the cellular level. CONCLUSIONS: Collectively, our results have demonstrated a novel mechanism by which cell-free CD34Exo mediates ischemic tissue repair via beneficial angiogenesis. Exosome-shuttled proangiogenic miRNAs may signify amplification of stem cell function and may explain the angiogenic and therapeutic benefits associated with CD34+ stem cell therapy.

Experimental and Computational Insight Into Human Mesenchymal Stem Cell Paracrine Signaling and Heterocellular Coupling Effects on Cardiac Contractility and Arrhythmogenicity.

RATIONALE: Myocardial delivery of human mesenchymal stem cells (hMSCs) is an emerging therapy for treating the failing heart. However, the relative effects of hMSC-mediated heterocellular coupling (HC) and paracrine signaling (PS) on human cardiac contractility and arrhythmogenicity remain unresolved. OBJECTIVE: The objective is to better understand hMSC PS and HC effects on human cardiac contractility and arrhythmogenicity by integrating experimental and computational approaches. METHODS AND RESULTS: Extending our previous hMSC-cardiomyocyte HC computational model, we incorporated experimentally calibrated hMSC PS effects on cardiomyocyte L-type calcium channel/sarcoendoplasmic reticulum calcium-ATPase activity and cardiac tissue fibrosis. Excitation-contraction simulations of hMSC PS-only and combined HC+PS effects on human cardiomyocytes were representative of human engineered cardiac tissue (hECT) contractile function measurements under matched experimental treatments. Model simulations and hECTs both demonstrated that hMSC-mediated effects were most pronounced under PS-only conditions, where developed force increased ≈4-fold compared with non-hMSC-supplemented controls during physiological 1-Hz pacing. Simulations predicted contractility of isolated healthy and ischemic adult human cardiomyocytes would be minimally sensitive to hMSC HC, driven primarily by PS. Dominance of hMSC PS was also revealed in simulations of fibrotic cardiac tissue, where hMSC PS protected from potential proarrhythmic effects of HC at various levels of engraftment. Finally, to study the nature of the hMSC paracrine effects on contractility, proteomic analysis of hECT/hMSC conditioned media predicted activation of PI3K/Akt signaling, a recognized target of both soluble and exosomal fractions of the hMSC secretome. Treating hECTs with exosome-enriched, but not exosome-depleted, fractions of the hMSC secretome recapitulated the effects observed with hMSC conditioned media on hECT-developed force and expression of calcium-handling genes (eg, SERCA2a, L-type calcium channel). CONCLUSIONS: Collectively, this integrated experimental and computational study helps unravel relative hMSC PS and HC effects on human cardiac contractility and arrhythmogenicity, and provides novel insight into the role of exosomes in hMSC paracrine-mediated effects on contractility.

Experimental, Systems, and Computational Approaches to Understanding the MicroRNA-Mediated Reparative Potential of Cardiac Progenitor Cell-Derived Exosomes From Pediatric Patients.

RATIONALE: Studies have demonstrated that exosomes can repair cardiac tissue post-myocardial infarction and recapitulate the benefits of cellular therapy. OBJECTIVE: We evaluated the role of donor age and hypoxia of human pediatric cardiac progenitor cell (CPC)-derived exosomes in a rat model of ischemia-reperfusion injury. METHODS AND RESULTS: Human CPCs from the right atrial appendages from children of different ages undergoing cardiac surgery for congenital heart defects were isolated and cultured under hypoxic or normoxic conditions. Exosomes were isolated from the culture-conditioned media and delivered to athymic rats after ischemia-reperfusion injury. Echocardiography at day 3 post-myocardial infarction suggested statistically improved function in neonatal hypoxic and neonatal normoxic groups compared with saline-treated controls. At 28 days post-myocardial infarction, exosomes derived from neonatal normoxia, neonatal hypoxia, infant hypoxia, and child hypoxia significantly improved cardiac function compared with those from saline-treated controls. Staining showed decreased fibrosis and improved angiogenesis in hypoxic groups compared with controls. Finally, using sequencing data, a computational model was generated to link microRNA levels to specific outcomes. CONCLUSIONS: CPC exosomes derived from neonates improved cardiac function independent of culture oxygen levels, whereas CPC exosomes from older children were not reparative unless subjected to hypoxic conditions. Cardiac functional improvements were associated with increased angiogenesis, reduced fibrosis, and improved hypertrophy, resulting in improved cardiac function; however, mechanisms for normoxic neonatal CPC exosomes improved function independent of those mechanisms. This is the first study of its kind demonstrating that donor age and oxygen content in the microenvironment significantly alter the efficacy of human CPC-derived exosomes.

Methodological Guidelines to Study Extracellular Vesicles.

Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, in this review, we will focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research.

Exosomes in Myocardial Repair: Advances and Challenges in the Development of Next-Generation Therapeutics.

Myocardial disease is a leading cause of morbidity and mortality worldwide. Given the limited regenerative capacity of the human heart following myocardial injury, stem cell-based therapies have emerged as a promising approach for improving cardiac repair and function. The discovery of extracellular vesicles including exosomes as a key component of the beneficial function of stem cells has generated hope for their use to advance cell-based regenerative therapies for cardiac repair. Exosomes secreted from stem cells are membranous bionanovesicles, naturally loaded with various proteins, lipids, and nucleic acids. They have been found to have anti-apoptotic, anti-fibrotic, as well as pro-angiogenic effects, all of which are crucial to restore function of the damaged myocardium. In this brief review, we will focus on the latest research and debates on cardiac repair and regenerative potential of exosomes from a variety of sources such as cardiac and non-cardiac stem and progenitor cells, somatic cells, and body fluids. We will also highlight important barriers involved in translating these findings into developing clinically suitable exosome-based therapies.

Exosomes and cardiac repair after myocardial infarction.

Myocardial infarction is a leading cause of death among all cardiovascular diseases. The analysis of molecular mechanisms by which the ischemic myocardium initiates repair and remodeling indicates that secreted soluble factors are key players in communication to local and distant tissues, such as bone marrow. Recently, actively secreted membrane vesicles, including exosomes, are being recognized as new candidates with important roles in intercellular and tissue-level communication. In this review, we critically examine the emerging role of exosomes in local and distant microcommunication mechanisms after myocardial infarction. A comprehensive understanding of the role of exosomes in cardiac repair after myocardial infarction could bridge a major gap in knowledge of the repair mechanism after myocardial injury.

Sonic hedgehog-modified human CD34+ cells preserve cardiac function after acute myocardial infarction.

RATIONALE: Ischemic cardiovascular disease represents one of the largest epidemics currently facing the aging population. Current literature has illustrated the efficacy of autologous, stem cell therapies as novel strategies for treating these disorders. The CD34+ hematopoetic stem cell has shown significant promise in addressing myocardial ischemia by promoting angiogenesis that helps preserve the functionality of ischemic myocardium. Unfortunately, both viability and angiogenic quality of autologous CD34+ cells decline with advanced age and diminished cardiovascular health. OBJECTIVE: To offset age- and health-related angiogenic declines in CD34+ cells, we explored whether the therapeutic efficacy of human CD34+ cells could be enhanced by augmenting their secretion of the known angiogenic factor, sonic hedgehog (Shh). METHODS AND RESULTS: When injected into the border zone of mice after acute myocardial infarction, Shh-modified CD34+ cells (CD34(Shh)) protected against ventricular dilation and cardiac functional declines associated with acute myocardial infarction. Treatment with CD34(Shh) also reduced infarct size and increased border zone capillary density compared with unmodified CD34 cells or cells transfected with the empty vector. CD34(Shh) primarily store and secrete Shh protein in exosomes and this storage process appears to be cell-type specific. In vitro analysis of exosomes derived from CD34(Shh) revealed that (1) exosomes transfer Shh protein to other cell types, and (2) exosomal transfer of functional Shh elicits induction of the canonical Shh signaling pathway in recipient cells. CONCLUSIONS: Exosome-mediated delivery of Shh to ischemic myocardium represents a major mechanism explaining the observed preservation of cardiac function in mice treated with CD34(Shh) cells.

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Exosomes from human CD34(+) stem cells mediate their proangiogenic paracrine activity.

RATIONALE: Transplantation of human CD34(+) stem cells to ischemic tissues has been associated with reduced angina, improved exercise time, and reduced amputation rates in phase 2 clinical trials and has been shown to induce neovascularization in preclinical models. Previous studies have suggested that paracrine factors secreted by these proangiogenic cells are responsible, at least in part, for the angiogenic effects induced by CD34(+) cell transplantation. OBJECTIVE: Our objective was to investigate the mechanism of CD34(+) stem cell-induced proangiogenic paracrine effects and to examine if exosomes, a component of paracrine secretion, are involved. METHODS AND RESULTS: Exosomes collected from the conditioned media of mobilized human CD34(+) cells had the characteristic size (40 to 90 nm; determined by dynamic light scattering), cup-shaped morphology (electron microscopy), expressed exosome-marker proteins CD63, phosphatidylserine (flow cytometry) and TSG101 (immunoblotting), besides expressing CD34(+) cell lineage marker protein, CD34. In vitro, CD34(+) exosomes replicated the angiogenic activity of CD34(+) cells by increasing endothelial cell viability, proliferation, and tube formation on Matrigel. In vivo, the CD34(+) exosomes stimulated angiogenesis in Matrigel plug and corneal assays. Interestingly, exosomes from CD34(+) cells but not from CD34(+) cell-depleted mononuclear cells had angiogenic activity. CONCLUSIONS: Our data demonstrate that human CD34(+) cells secrete exosomes that have independent angiogenic activity both in vitro and in vivo. CD34(+) exosomes may represent a significant component of the paracrine effect of progenitor cell transplantation for therapeutic angiogenesis.