Susmita Sahoo

Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles

Secreted membrane-enclosed vesicles, collectively called extracellular vesicles (EVs), which include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies and other EV subsets, encompass a very rapidly growing scientific field in biology and medicine. Importantly, it is currently technically challenging to obtain a totally pure EV fraction free from non-vesicular components for functional studies, and therefore there is a need to establish guidelines for analyses of these vesicles and reporting of scientific studies on EV biology. Here, the International Society for Extracellular Vesicles (ISEV) provides researchers with a minimal set of biochemical, biophysical and functional standards that should be used to attribute any specific biological cargo or functions to EVs.

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.

EVpedia: a community web portal for extracellular vesicles research

MOTIVATION Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. RESULTS We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. AVAILABILITY AND IMPLEMENTATION The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey

Extracellular vesicles (EVs) represent an important mode of intercellular communication. Research in this field has grown rapidly in the last few years, and there is a plethora of techniques for the isolation and characterization of EVs, many of which are poorly standardized. EVs are heterogeneous in size, origin and molecular constituents, with considerable overlap in size and phenotype between different populations of EVs. Little is known about current practices for the isolation, purification and characterization of EVs. We report here the first large, detailed survey of current worldwide practices for the isolation and characterization of EVs. Conditioned cell culture media was the most widely used material (83%). Ultracentrifugation remains the most commonly used isolation method (81%) with 59% of respondents use a combination of methods. Only 9% of respondents used only 1 characterization method, with others using 2 or more methods. Sample volume, sample type and downstream application all influenced the isolation and characterization techniques employed.

Exosomes and exosomal miRNAs in cardiovascular protection and repair

Cell-cell communication between cardiac and vascular cells and from stem and progenitor cells to differentiated cardiovascular cells is both an important and complex process, achieved through a diversity of mechanisms that have an impact on cardiovascular biology, disease and therapeutics. In recent years, evidence has accumulated suggesting that extracellular vesicles (EVs) are a new system of intercellular communication. EVs of different sizes are produced via different biogenesis pathways and have been shown to be released and taken up by most of known cell types, including heart and vascular cells, and stem and progenitor cells. This review will focus on exosomes, the smallest EVs (up to 100nm in diameter) identified so far. Cells can package cargoes consisting of selective lipids, proteins and RNA in exosomes and such cargoes can be shipped to recipient cells, inducing expressional and functional changes. This review focuses on exosomes and microRNAs in the context of cardiovascular disease and repair. We will describe exosome biogenesis and cargo formation and discuss the available information on in vitro and in vivo exosomes-based cell-to-cell communication relevant to cardiovascular science. The methods used in exosome research will be also described. Finally, we will address the promise of exosomes as clinical biomarkers and their impact as a biomedical tool in stem cell-based cardiovascular therapeutics.

Induction of Autophagy by Cystatin C: A Mechanism That Protects Murine Primary Cortical Neurons and Neuronal Cell Lines

Cystatin C (CysC) expression in the brain is elevated in human patients with epilepsy, in animal models of neurodegenerative conditions, and in response to injury, but whether up-regulated CysC expression is a manifestation of neurodegeneration or a cellular repair response is not understood. This study demonstrates that human CysC is neuroprotective in cultures exposed to cytotoxic challenges, including nutritional-deprivation, colchicine, staurosporine, and oxidative stress. While CysC is a cysteine protease inhibitor, cathepsin B inhibition was not required for the neuroprotective action of CysC. Cells responded to CysC by inducing fully functional autophagy via the mTOR pathway, leading to enhanced proteolytic clearance of autophagy substrates by lysosomes. Neuroprotective effects of CysC were prevented by inhibiting autophagy with beclin 1 siRNA or 3-methyladenine. Our findings show that CysC plays a protective role under conditions of neuronal challenge by inducing autophagy via mTOR inhibition and are consistent with CysC being neuroprotective in neurodegenerative diseases. Thus, modulation of CysC expression has therapeutic implications for stroke, Alzheimers disease, and other neurodegenerative disorders.

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.

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.

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.