Discovery Of Extracellular Vesicles Research Articles

Proteomic differences between extracellular vesicles and extracellular vesicle-depleted excretory/secretory products of barber’s pole worm

BackgroundComponents of excretory/secretory products (ESPs) of helminths have been proposed as vaccine targets and shown to play a role in modulating host immune responses for decades. Such research interest is further increased by the discovery of extracellular vesicles (EVs) in the ESPs of parasitic worms. Although efforts have been made to reveal the cargos of EVs, little is known about the proteomic differences between EVs and canonical ESPs released by parasitic worms from animals.MethodsThe total ESPs of Haemonchuscontortus (barber’s pole worm) were obtained by short-term in vitro culturing of young adult worms, and small EVs were isolated from ESPs using an ultracentrifugation method. Data-dependent acquisition (DDA) label-free Nano-LC–MS/MS was used to quantify the proteomic difference between small EVs and EV-depleted ESPs of H.contortus. Functional annotation and enrichment of the differential proteins were performed regarding cellular components, molecular functions, pathways, and/or biological processes.ResultsA total of 1697 proteins were identified in small EVs and EV-depleted ESPs of H.contortus adult worms, with 706 unique proteins detected in the former and 597 unique proteins in the latter. It was revealed that proteins in small EVs are dominantly cytoplasmic, whereas proteins in EV-depleted ESPs are mainly extracellular; canonical ESPs such as proteases and small GTPases were abundantly detected in small EVs, and SCP/TAP-, DUF-, and GLOBIN domain-containing proteins were mainly found in EV-depleted ESPs. Compared with well-characterised proteins in small EVs, about 50% of the proteins detected in EV-depleted ESPs were poorly characterised.ConclusionsThere are remarkable differences between small EVs and EV-depleted ESPs of H.contortus in terms of protein composition. Immune modulatory effects caused by nematode ESPs are possibly contributed mainly by the proteins in small EVs.Graphical

Fibroblast-derived extracellular vesicles as trackable efficient transporters of an experimental nanodrug with fibrotic heart and lung targeting.

The discovery of extracellular vesicles (EVs) as efficient exogenous biotransporters of therapeutic agents into cells across biological membranes is an exciting emerging field. Especially the potential of EVs as targeted delivery systems for diseases with selective treatments, such as fibrosis, whose treatment causes side effects in other organs not involved in the disease. Methods: In this study, we collected embryonic fibroblast-derived EVs from two different centrifugation fractions, 10 K g and 100 K g fractions from a NIH-3T3 cell line loaded with an experimental drug. Mice with fibrotic hearts and lungs were obtained by administration of angiotensin II. We generated fluorescent EVs and bioluminescent drug to observe their accumulation by colocalization of their signals in fibrotic heart and lung. The biodistribution of the drug in various organs was obtained by detecting the Au present in the drug nanostructure. Results: The drug-loaded EVs successfully reduced fibrosis in pathological fibroblasts in vitro, and modified the biodistribution of the experimental drug, enabling it to reach the target organs in vivo. We described the pre-analytical characteristics of EVs related to physical variables, culture and harvesting conditions, crucial for their in vivo application as nanotransporters using a previously validated protein-based antifibrotic drug. The results showed the colocalization of EVs and the experimental drug in vivo and ex vivo and the efficient reduction of fibrosis in vitro. This work demonstrates that 10K-EVs and 100K-EVs derived from fibroblasts can act as effective biotransporters for targeted drug delivery to profibrotic fibroblasts, lungs, or heart. Conclusion: We observed that fibroblast-derived 10K-EVs and 100K-EVs are useful biotransporters encapsulating a new generation drug leading to a reduction of fibrosis in profibrotic fibroblasts in vitro. In addition, drug containing EVs were shown to reach fibrotic heart and lungs in vivo, enhancing free drug biodistribution.

Unlocking Exosome-Based Theragnostic Signatures: Deciphering Secrets of Ovarian Cancer Metastasis.

Ovarian cancer (OC) is a common gynecological cancer worldwide. Unfortunately, the lack of early detection methods translates into a substantial cohort of women grappling with the pressing health crisis. The discovery of extracellular vesicles (EVs) (their major subpopulation exosomes, microvesicles, and apoptotic bodies) has provided new insights into the understanding of cancer. Exosomes, a subpopulation of EVs, play a crucial role in cellular communication and reflect the cellular status under both healthy and pathological conditions. Tumor-derived exosomes (TEXs) dynamically influence ovarian cancer progression by regulating uncontrolled cell growth, immune suppression, angiogenesis, metastasis, and the development of drug and therapeutic resistance. In the field of OC diagnostics, TEXs offer potential biomarkers in various body fluids. On the other hand, exosomes have also shown promising abilities to cure ovarian cancer. In this review, we address the interlink between exosomes and ovarian cancer and explore their theragnostic signature. Finally, we highlight future directions of exosome-based ovarian cancer research.

Environmental and Lifestyle Cancer Risk Factors: Shaping Extracellular Vesicle OncomiRs and Paving the Path to Cancer Development.

Intercellular communication has been transformed by the discovery of extracellular vesicles (EVs) and their cargo, including microRNAs (miRNAs), which play crucial roles in intercellular signaling. These EVs were previously disregarded as cellular debris but are now recognized as vital mediators of biological information transfer between cells. Furthermore, they respond not only to internal stimuli but also to environmental and lifestyle factors. Identifying EV-borne oncomiRs, a subset of miRNAs implicated in cancer development, could revolutionize our understanding of how environmental and lifestyle exposures contribute to oncogenesis. To investigate this, we studied the plasma levels of EV-borne oncomiRs in a population of 673 women and 238 men with a body mass index > 25 kg/m2 (SPHERE population). The top fifty oncomiRs associated with the three most common cancers in women (breast, colorectal, and lung carcinomas) and men (lung, prostate, and colorectal carcinomas) were selected from the OncomiR database. Only oncomiRs expressed in more than 20% of the population were considered for statistical analysis. Using a Multivariate Adaptive Regression Splines (MARS) model, we explored the interactions between environmental/lifestyle exposures and EV oncomiRs to develop optimized predictor combinations for each EV oncomiR. This innovative approach allowed us to better understand miRNA regulation in response to multiple environmental and lifestyle influences. By uncovering non-linear relationships among variables, we gained valuable insights into the complexity of miRNA regulatory networks. Ultimately, this research paves the way for comprehensive exposome studies in the future.

New signaling kid on the block in the endocrine system: the role of extracellular vesicles.

In recent years, there has been a growing interest in the role of Extracellular vesicles (EVs) in both normal and pathological physiology. These natural nanoparticles are now recognized as a novel mechanism for intercellular communication, allowing cells to exchange of biologically active molecules such as microRNAs (miRNAs). As is well acknowledged, the endocrine system regulates bodily operations through the emission of various hormones. Although the discovery of EVs took place approximately 80 years later than that of hormones, circulating EVs have attracted considerable interest and are expected to be the frontier in the endocrine system. Interestingly, the interplay between hormones and EVs is a complex phenomenon that involves both synergistic and antagonistic effects. Moreover, EVs facilitate communication between endocrine cells and contain miRNAs that may serve as valuable biomarkers for diagnosis and prognosis. This review aims to provide an overview of current research on physiological and pathological secretion of EVs from endocrine organs or tissues. Additionally, we examine the essential relationship between hormones and EVs in the endocrine system.

Concise review: Current understanding of extracellular vesicles to treat neuropathic pain.

Extracellular vesicles (EVs) including exosomes are vesicular vesicles with phospholipid bilayer implicated in many cellular interactions and have the ability to transfer multiple types of cargo to cells. It has been found that EVs can package various molecules including proteins and nucleic acids (DNA, mRNA, and noncoding RNA). The discovery of EVs as carriers of proteins and various forms of RNA, such as microRNAs (miRNA) and long noncoding RNAs (lncRNA), has raised great interest in the field of drug delivery. Despite the underlying mechanisms of neuropathic pain being unclear, it has been shown that uncontrolled glial cell activation and the neuroinflammation response to noxious stimulation are important in the emergence and maintenance of neuropathic pain. Many studies have demonstrated a role for noncoding RNAs in the pathogenesis of neuropathic pain and EVs may offer possibilities as carriers of noncoding RNAs for potential in neuropathic pain treatment. In this article, the origins and clinical application of EVs and the mechanism of neuropathic pain development are briefly introduced. Furthermore, we demonstrate the therapeutic roles of EVs in neuropathic pain and that this involve vesicular regulation of glial cell activation and neuroinflammation.

Extracellular Vesicles in Mycobacteria and Tuberculosis.

Tuberculosis (TB) remains a public health issue causing millions of infections every year. Of these, about 15% ultimately result in death. Efforts to control TB include development of new and more effective vaccines, novel and more effective drug treatments, and new diagnostics that test for both latent TB Infection and TB disease. All of these areas of research benefit from a good understanding of the physiology of Mycobacterium tuberculosis (Mtb), the primary causative agent of TB. Mtb secreted protein antigens have been the focus of vaccine and diagnosis research for the past century. Recently, the discovery of extracellular vesicles (EVs) as an important source of secreted antigens in Mtb has gained attention. Similarly, the discovery that host EVs can carry Mtb products during in vitro and in vivo infection has spiked interest because of its potential use in blood-based diagnostics. Despite advances in understanding the content of Mtb and Mtb-infected host extracellular vesicles, our understanding on the biogenesis and role of Mtb and host extracellular vesicles during Mtb infection is still nascent. Here, we explore the current literature on extracellular vesicles regarding Mtb, discuss the host and Mtb extracellular vesicles as distinct entities, and discuss current gaps in the field.

EXTRACELLULAR VESICLE-ASSOCIATED MIRNA CARGO COULD EXPLAIN REGENERATIVE CAPACITIES OF BLOOD-DERIVED EXTRACELLULAR VESICLES ON PRIMARY OSTEOARTHRITIC CHONDROCYTES

Purpose: Regenerative medicine increasingly focuses on blood-derived products for osteoarthritis therapy. Frequently, citrate-anti-coagulated platelet-rich plasma (CPRP) is intra-articularly injected into diseased joints, however, cell-free alternatives such as hyperacute serum (hypACT) are under development. Mechanisms of action of blood products are still poorly understood. The discovery of extracellular vesicles (EVs) in blood and EV-associated cargo molecules such as miRNAs opened up new levels of complexity in understanding the therapeutic potential of blood products.

Extracellular vesicles and immune response during pregnancy: A balancing act.

The mechanisms underlying maternal tolerance of the semi- or fully-allogeneic fetus are intensely investigated. Across gestation, feto-placental antigens interact with the maternal immune system locally within the trophoblast-decidual interface and distantly through shed cells and soluble molecules that interact with maternal secondary lymphoid tissues. The discovery of extracellular vesicles (EVs) as local or systemic carriers of antigens and immune-regulatory molecules has added a new dimension to our understanding of immune modulation prior to implantation, during trophoblast invasion, and throughout the course of pregnancy. New data on immune-regulatory molecules, located on EVs or within their cargo, suggest a role for EVs in negotiating immune tolerance during gestation. Lessons from the field of transplant immunology also shed light on possible interactions between feto-placentally derived EVs and maternal lymphoid tissues. These insights illuminate a potential role for EVs in major obstetrical disorders. This review provides updated information on intensely studied, pregnancy-related EVs, their cargo molecules, and patterns of fetal-placental-maternal trafficking, highlighting potential immune pathways that might underlie immune suppression or activation in gestational health and disease. Our summary also underscores the likely need to broaden the definition of the maternal-fetal interface to systemic maternal immune tissues that might interact with circulating EVs.

Multiple Facets of Plant-Microbiome Associations in Unlocking the Communication Paradigm through Extracellular Vesicles.

Communication among different species across kingdoms occurs through a chain of regulatory molecules that are transferred around cellular boundaries. These molecules are also crucial for defense, virulence, and pathogenesis. In the past, the transport of proteins in long distance communication was observed, but in the present era, the discovery of extracellular vesicles (EVs) has changed our understanding of molecular communication. EVs are not only involved in cell signaling and immunity but also can transfer information by sRNAs, forming a basis for interactions among a wide variety of organisms. Despite extensive research on EVs in other areas, their role in communication between plants and the plant microbiome has been lacking. EVs are potentially involved in protein trafficking along with the transport of lipids and nucleic acids. Interactions between hosts and their microbiomes may also be mediated by EVs, which can be involved in stress responses, immune surveillance and defense, virulence, and signaling, along with many metabolic activities within plant microbiomes. In this review, we have focused on recent information about the role of EVs and the molecules they transport between hosts and microbes. The connection between biofilms and the generation of EVs is also considered. These findings enhance our knowledge about plant-microbiome interactions in terms of immunity and virulence and challenge the conventional viewpoint of inter-kingdom signaling.

Functional repertoire of EV-associated miRNA profiles after lipoprotein depletion via ultracentrifugation and size exclusion chromatography from autologous blood products

Cartilage breakdown, inflammation and pain are hallmark symptoms of osteoarthritis, and autologous blood products such as citrate-anticoagulated platelet-rich plasma (CPRP) or hyperacute serum (hypACT) have been developed as a regenerative approach to rebuild cartilage, inhibit inflammation and reduce pain. However, mechanisms of action of these blood derivatives are still not fully understood, in part due to the large number of components present in these medical products. In addition, the discovery of extracellular vesicles (EVs) and their involvement in intercellular communication mediated by cargo molecules like microRNAs (miRNAs) opened up a whole new level of complexity in understanding blood products. In this study we focused on the development of an isolation protocol for EVs from CPRP and hypACT that can also deplete lipoproteins, which are often co-isolated in EV research due to shared physical properties. Several isolation methods were compared in terms of particle yield from CPRP and hypACT. To gain insights into the functional repertoire conveyed via EV-associated miRNAs, we performed functional enrichment analysis and identified NFκB signaling strongly targeted by CPRP EV miRNAs, whereas hypACT EV miRNAs affect IL6- and TGFβ/SMAD signaling.

Inhibition of extracellular vesicle pathway using neutral sphingomyelinase inhibitors as a neuroprotective treatment for brain injury.

Traumatic brain injury is a sudden trauma or blow on the head, and severe traumatic brain injury is a major cause of death and disability worldwide. The acute and chronic consequences following traumatic brain injury can lead to progressive secondary neurodegenerative changes and cognitive dysfunction. To date, there is no effective pharmaceutical products for the treatment to reduce secondary damage after brain injury. The discovery of extracellular vesicles has attracted considerable scientific attention due to their role in cell-to-cell communication. Extracellular vesicles have shown their potential to carry not only biological molecules but also as a drug delivery vehicle. As a carrier of molecular information, extracellular vesicles have been involved in physiological functions as well as in the modulation of immune responses. Here, we aim to provide new insights into the contrasting role of extracellular vesicles in the propagation of inflammatory responses after brain injury. As a carrier of pro-inflammatory molecules, their role as functional mediators in the pathophysiology of brain injury is discussed, addressing the inhibition of the extracellular vesicle pathway as an anti-inflammatory or neuroprotective approach to improve the outcome of both acute and chronic inflammation following brain injury. Here, we summarize therapeutic strategies to diminish the risk the neurodegeneration post brain injury and propose that neutral sphingomyelinase inhibitors could be used as potentially useful therapeutic agents for the treatment of brain injury associated neuroinflammation.

Extracellular Vesicles as Mediators of Cellular Cross Talk in the Lung Microenvironment.

The human lung is a complex tissue subdivided into several regions that differ in size, function, and resident cell types. Despite years of intensive research, we still do not fully understand the cross talk between these different regions and diverse cell populations in the lung and how this is altered in the development of chronic respiratory disease. The discovery of extracellular vesicles (EVs), small membrane vesicles released from cells for intercellular communication, has added another layer of complexity to cellular cross talk in the complex lung microenvironment. EVs from patients with chronic obstructive pulmonary disease, asthma, or sarcoidosis have been shown to carry microRNAs, proteins, and lipids that may contribute to inflammation or tissue degeneration. Here, we summarize the contribution of these small vesicles in the interplay of several different cell types in the lung microenvironment, with a focus on the development of chronic respiratory diseases. Although there are already many studies demonstrating the adverse effects of EVs in the diseased lung, we still have substantial knowledge gaps regarding the concrete role of EV involvement in lung disease, which should be addressed in future studies.

Extracellular Vesicles Function as Bioactive Molecular Transmitters in the Mammalian Oviduct: An Inspiration for Optimizing in Vitro Culture Systems and Improving Delivery of Exogenous Nucleic Acids during Preimplantation Embryonic Development.

Two technologies, in vitro culture and exogenous gene introduction, constitute cornerstones of producing transgenic animals. Although in vitro embryo production techniques can bypass the oviduct during early development, such embryos are inferior to their naturally produced counterparts. In addition, preimplantation embryos are resistant to the uptake of exogenous genetic material. These factors restrict the production of transgenic animals. The discovery of extracellular vesicles (EVs) was a milestone in the study of intercellular signal communication. EVs in the oviduct, known as oviductosomes (OVS), are versatile delivery tools during maternal–embryo communication. In this review, we discuss the important roles of OVS in these interactions and the feasibility of using them as tools for transferring exogenous nucleic acids during early development. We hypothesize that further accurate characterization of OVS cargoes and functions will open new horizons for research on maternal–embryo interactions and enhance the production of transgenic animals.

Answers to naysayers regarding microbial extracellular vesicles.

It is now over 30 years since the discovery of extracellular vesicles (EVs) in Gram-negative bacteria. However, for cell-walled microbes such as fungi, mycobacteria and Gram-positive bacteria it was thought that EV release would be impossible, since such structures were not believed to cross the thick cell wall. This notion was disproven 10 years ago with the discovery of EVs in fungi, mycobacteria, and gram-positive bacteria. Today, EVs have been described in practically every species tested, ranging from Fungi through Bacteria and Archaea, suggesting that EVs are a feature of every living cell. However, there continues to be skepticism in some quarters regarding EV release and their biological significance. In this review, we list doubts that have been verbalized to us and provide answers to counter them. In our opinion, there is no doubt as to existence and physiological function of EVs and we take this opportunity to highlight the most pressing topics in our understanding of the biological processes underlying these structures.

Extracellular Vesicles and Cell-Cell Communication in the Cornea.

ABSTRACTOne question that has intrigued cell biologists for many years is, “How do cells interact to influence one another's activity?” The discovery of extracellular vesicles (EVs) and the fact that they carry cargo, which directs cells to undergo changes in morphology and gene expression, has revolutionized this field of research. Little is known regarding the role of EVs in the cornea; however, we have demonstrated that EVs isolated from corneal epithelial cells direct corneal keratocytes to initiate fibrosis. Intriguingly, our data suggest that EVs do not penetrate epithelial basement membrane (BM), perhaps providing a mechanism explaining the importance of BM in the lack of scarring in scrape wounds. Since over 100‐million people worldwide suffer from visual impairment as a result of corneal scarring, the role of EVs may be vital to understanding the mechanisms of wound repair. Therefore, we investigated EVs in ex vivo and in vivo‐like three‐dimensional cultures of human corneal cells using transmission electron microscopy. Some of the major findings were all three major cell types (epithelial, fibroblast, and endothelial cells) appear to release EVs, EVs can be identified using TEM, and EVs appeared to be involved in cell–cell communication. Interestingly, while our previous publication suggests that EVs do not penetrate the epithelial BM, it appears that EVs penetrate the much thicker endothelial BM (Descemet's membrane). These findings indicate the huge potential of EV research in the cornea and wound healing, and suggest that during homeostasis the endothelium and stromal cells are in communication. Anat Rec, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Identification of extracellular vesicles and characterization of miRNA expression profiles in human blastocoel fluid

In this study, for the first time, we demonstrated the presence of microRNAs and extracellular vesicles in human blastocoel fluid. The bioinformatic and comparative analyses identified the biological function of blastocoel fluid microRNAs and suggested a potential role inside the human blastocyst. We found 89 microRNAs, expressed at different levels, able to regulate critical signaling pathways controlling embryo development, such as pluripotency, cell reprogramming, epigenetic modifications, intercellular communication, cell adhesion and cell fate. Blastocoel fluid microRNAs reflect the miRNome of embryonic cells and their presence, associated with the discovery of extracellular vesicles, inside blastocoel fluid, strongly suggests their important role in mediating cell communication among blastocyst cells. Their characterization is important to better understand the earliest stages of embryogenesis and the complex circuits regulating pluripotency. Moreover, blastocoel fluid microRNA profiles could be influenced by blastocyst quality, therefore, microRNAs might be used to assess embryo potential in IVF cycles.

Extracellular Vesicles and the Promise of Continuous Liquid Biopsies.

The rapid and accurate diagnosis of patients with minimally invasive procedures was once only found in science fiction. However, the discovery of extracellular vesicles (EVs) and their near ubiquity in body fluids, coupled with the advent of inexpensive next generation sequencing techniques and EV purification protocols, promises to make science fiction a reality. Purifying and sequencing the RNA content of EV from routine blood draws and urine samples are likely to enable pathologists and physicians to diagnose and track the progress of diseases in many inaccessible tissues in the near future. Here we present the evolutionary background of EV, summarize the biology of EV formation and cargo selection, and discuss the current barriers to making continuous liquid biopsies through the use of EV a science reality.

Abstract 3150: Analysis of the extracellular vesicles content present in the plasma of patients with head and neck squamous cell carcinoma for identification of molecular markers for treatment response

Abstract Previous data showed that induction chemotherapy (IC) with cisplatin and paclitaxel followed by chemoradiotherapy (CRT) based on cisplatin is safe, well tolerated and improves overall survival for patients with locally advanced head and neck squamous cell carcinoma (HNSCC) (stage III -IV). However, approximately 30% of these patients have incomplete response to IC and CRT and there is no clinical marker able to segregate these patients from those that respond to the treatment. The recent discovery of extracellular vesicles (EVs) in different body fluids carrying proteins and nucleic acids may indicate an alternative route for new specific markers present in the plasma of patients with HNSCC. In this study, Cholera toxin B chain (CTB) and Annexin V (AV) which respectively binds GM1 ganglioside and phosphatidylserine, were used to isolate EVs from plasma of HNSCC patients that respond and not respond to IC and CRT. The proteins in the vesicles were identified using antibody array through a pool of 6 responders’ patients (R) in comparison to 6 patients with incomplete response (IR). A total of 327 proteins were identified into CTB-EVs, from which 54 and 20 proteins were exclusive to R and IR patients, respectively. Beside this, 271 proteins were identified into AV-EVs, from which 20 proteins were present in R patients and other 20 proteins were present only in IR ones. A preliminary analysis of this data revealed that HNSCC resistant patients have elevated levels of TGFβ3 in CTB-EVs while patients with incomplete response have elevated levels of HSP90AB1 in AV-EVs. This study demonstrated the successful use of CTB and AV to isolate circulating plasma EVs for biomarker discovery in HNSCC and represents a novel technology that can be applied to segregate patients that may respond or not to IC or CRT. Citation Format: Dorival Mendes Rodrigues-Junior, Soon Sim Tan, Sai Kiang Lim, N Gopalakrishna Iyer, Andre Luiz Vettore. Analysis of the extracellular vesicles content present in the plasma of patients with head and neck squamous cell carcinoma for identification of molecular markers for treatment response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3150.

Clinical Application of Human Urinary Extracellular Vesicles in Kidney and Urologic Diseases.

Extracellular vesicles (EVs) have been isolated in different body fluids, including urine. The cargo of urinary EVs is composed of nucleic acids and proteins reflecting the physiological and possibly pathophysiological state of cells lining the nephron and the urinary tract. Urinary EVs have been confirmed to contain low amounts of various types of RNA that play a role in intercellular communication by transferring genetic information. This communication through EV RNAs includes both continuation of normal physiological processes and conditioning in disease mechanisms. Although proteins included in urinary EVs represent only 3% of the whole-urine proteome, urinary EVs can influence cells in the renal epithelia not only by delivering RNA cargo, but also by delivering a wide range of proteins. Since urine is a readily available biofluid, the discovery of EVs has opened a new field of biomarker research. The potential use of urinary EV RNAs and proteins as diagnostic biomarkers for various kidney and urologic diseases is currently being explored. Here, we review recent studies that deal in identifying biomarker candidates for human kidney and urologic diseases using urinary EVs and might help to understand the pathophysiology.