Extracellular Vesicles Biology Research Articles

Glucocerebrosidase reduces the spread of protein aggregation in a Drosophila melanogaster model of neurodegeneration by regulating proteins trafficked by extracellular vesicles.

Abnormal protein aggregation within neurons is a key pathologic feature of Parkinson’s disease (PD). The spread of brain protein aggregates is associated with clinical disease progression, but how this occurs remains unclear. Mutations in glucosidase, beta acid 1 (GBA), which encodes glucocerebrosidase (GCase), are the most penetrant common genetic risk factor for PD and dementia with Lewy bodies and associate with faster disease progression. To explore how GBA mutations influence pathogenesis, we previously created a Drosophila model of GBA deficiency (Gba1b) that manifests neurodegeneration and accelerated protein aggregation. Proteomic analysis of Gba1b mutants revealed dysregulation of proteins involved in extracellular vesicle (EV) biology, and we found altered protein composition of EVs from Gba1b mutants. Accordingly, we hypothesized that GBA may influence pathogenic protein aggregate spread via EVs. We found that accumulation of ubiquitinated proteins and Ref(2)P, Drosophila homologue of mammalian p62, were reduced in muscle and brain tissue of Gba1b flies by ectopic expression of wildtype GCase in muscle. Neuronal GCase expression also rescued protein aggregation both cell-autonomously in brain and non-cell-autonomously in muscle. Muscle-specific GBA expression reduced the elevated levels of EV-intrinsic proteins and Ref(2)P found in EVs from Gba1b flies. Perturbing EV biogenesis through neutral sphingomyelinase (nSMase), an enzyme important for EV release and ceramide metabolism, enhanced protein aggregation when knocked down in muscle, but did not modify Gba1b mutant protein aggregation when knocked down in neurons. Lipidomic analysis of nSMase knockdown on ceramide and glucosylceramide levels suggested that Gba1b mutant protein aggregation may depend on relative depletion of specific ceramide species often enriched in EVs. Finally, we identified ectopically expressed GCase within isolated EVs. Together, our findings suggest that GCase deficiency promotes accelerated protein aggregate spread between cells and tissues via dysregulated EVs, and EV-mediated trafficking of GCase may partially account for the reduction in aggregate spread.

Landscape of extracellular vesicles in the tumour microenvironment: Interactions with stromal cells and with non-cell components, and impacts on metabolic reprogramming, horizontal transfer of neoplastic traits, and the emergence of therapeutic resistance.

Extracellular vesicles (EVs) are increasingly recognised as a pivotal player in cell-cell communication, an attribute of EVs that derives from their ability to transport bioactive cargoes between cells, resulting in complex intercellular signalling mediated by EVs, which occurs under both physiological and pathological conditions. In the context of cancer, recent studies have demonstrated the versatile and crucial roles of EVs in the tumour microenvironment (TME). Here, we revisit EV biology, and focus on EV-mediated interactions between cancer cells and stromal cells, including fibroblasts, immune cells, endothelial cells and neurons. In addition, we focus on recent reports indicating interactions between EVs and non-cell constituents within the TME, including the extracellular matrix. We also review and summarise the intricate cancer-associated network modulated by EVs, which promotes metabolic reprogramming, horizontal transfer of neoplastic traits, and therapeutic resistance in the TME. We aim to provide a comprehensive and updated landscape of EVs in the TME, focusing on oncogenesis, cancer progression and therapeutic resistance, together with our future perspectives on the field.

Polarized cells display asymmetric release of extracellular vesicles.

Extracellular vesicles (EVs), a broad term for the lipid microparticles known as microvesicles and exosomes, are discharged by cells into their surrounding space. Microvesicles are discharged upon outward plasma membrane budding, while exosomes are secreted after multivesicular body (MVB) fusion with the plasma membrane. The majority of information regarding EV biology comes from studies performed in non-polarized cells. Here we characterize EV release in polarized cells. We found a substantial asymmetry in the number and composition of EVs produced and released from the apical membrane of epithelial cells as compared to the basolateral membrane. We showed that the quantitative difference is related to the polarized distribution of two phosphoinositide species between the two cell surfaces and that the peculiar biochemical composition of resultant EVs reflects their site of origin. In particular, apical and basolateral exosomes may derive from distinct classes of MVBs originating from and fusing with the same plasma membrane. We identify VAMP8/Endobrevin as a regulator of the basolateral release of exosomes, whereas the mechanism responsible for apical EV release requires further study.

Extracellular vesicles versus synthetic nanoparticles for drug delivery.

Cell-released biological nanoparticles, including extracellular vesicles (EVs), are emerging drug carriers with high complexity. EV-based drug delivery has the potential to efficiently exploit intrinsic mechanisms for molecular transport in the body. Integrating the expanding knowledge of EV biology and manufacturing with clinical insights from synthetic nanoparticles is likely to substantially advance the field of drug delivery.

Extracellular vesicle-shuttled miRNAs: a critical appraisal of their potential as nano-diagnostics and nano-therapeutics in type 2 diabetes mellitus and its cardiovascular complications.

Type 2 diabetes mellitus (T2DM) is a complex multifactorial disease causing the development of a large range of cardiovascular (CV) complications. Lifestyle changes and pharmacological therapies only partially halt T2DM progression, and existing drugs are unable to completely suppress the increased CV risk of T2DM patients. Extracellular vesicles (EV)s are membrane-coated nanoparticles released by virtually all living cells and are emerging as novel mediators of T2DM and its CV complications. As a matter of fact, several preclinical models suggest a key involvement of EVs in the initiation and/or progression of insulin resistance, β-cell dysfunction, diabetic dyslipidaemia, atherosclerosis, and other T2DM complications. In addition, preliminary findings also suggest that EV-associated molecular cargo, and in particular the miRNA repertoire, may provide with useful diagnostic and/or prognostic information for the management of T2DM. Here, we review the latest findings showing that EV biology is altered during the entire trajectory of T2DM, i.e. from diagnosis to development of CV complications. We also critically highlight the potential of this emerging research field, by describing both preclinical and clinical observations, and the limitations that must be overcome to translate the preclinical findings into the development of EV-based nano-diagnostic and/or nano-therapeutic tools. Finally, we summarize how two lifestyle changes known to prevent or limit T2DM, i.e. diet and exercise, affect EV number and composition, with a focus on the possible role of EVs contained in food in shaping metabolic responses, a promising approach still in its infancy.

Lessons Learned from Studying Histoplasma capsulatum Extracellular Vesicles.

Histoplasma capsulatum is a major endemic mycosis. Our laboratories have demonstrated that H. capsulatum produces extracellular vesicles (EV) that are loaded with diverse compounds that influence virulence. We have further shown that H. capsulatum dynamically regulates the loading and release of fungal EV in response to stimuli and growth conditions. This chapter details the current knowledge of EV biology in H. capsulatum and the impact of this information on our understanding of this important process that is closely linked to pathogenesis.

Extracellular Vesicles and Glycosylation.

Extracellular vesicles (EVs), a generic term for any vesicles or particles that are released from cells, play an important role in modulating numerous biological and pathological events, including development, differentiation, aging, thrombus formation, immune responses, neurodegenerative diseases, and tumor progression. During the biogenesis of EVs, they encapsulate biologically active macromolecules (i.e., nucleotides and proteins) and transmit signals for delivering them to neighboring or cells that are located some distance away. In contrast, there are receptor molecules on the surface of EVs that function to mediate EV-to-cell and EV-to-matrix interactions. A growing body of evidence indicates that the EV surface is heavily modified with glycans, the function of which is to regulate the biogenesis and extracellular behaviors of EVs. In this chapter, we introduce the current status of our knowledge concerning EV glycosylation and discuss how it influences EV biology, highlighting the potential roles of EV glycans in clinical applications.

Extracellular Vesicles in Neurological Disorders.

The role of extracellular vesicles (EVs) in the central nervous system, and in particular the brain, is a rapidly growing research area. Importantly, the role for EVs in the nervous system spans from early development through to old age, with EVs being associated with several different neurological disorders. To date,researchers have been studying the function of EVs in the nervous system in three major areas: (i) the role of EVs in promoting disease pathways, (ii) the ability of EVs to be used asa diagnostic tool to identify cellular changes in the nervous system, and (iii) the potential use of EVs as therapeutic tools for the delivery of biomolecules or drugs to the nervous system. In each of these settings the analysis and use of EVs performs a different function, highlighting the breadth of areas in which the EV field is applicable. A key aspect of EV biology is the ability of vesicles to cross biological barriers, in particular the blood brain barrier. This allows for the measurement of serum EVs that contain information about cells in the brain, or alternatively, allows for the delivery of biomolecules that are packaged within EVs for therapeutic use.

Effects of Chronic Kidney Disease and Uremic Toxins on Extracellular Vesicle Biology.

Vascular calcification (VC) is a cardiovascular complication associated with a high mortality rate, especially in patients with diabetes, atherosclerosis or chronic kidney disease (CKD). In CKD patients, VC is associated with the accumulation of uremic toxins, such as indoxyl sulphate or inorganic phosphate, which can have a major impact in vascular remodeling. During VC, vascular smooth muscle cells (VSMCs) undergo an osteogenic switch and secrete extracellular vesicles (EVs) that are heterogeneous in terms of their origin and composition. Under physiological conditions, EVs are involved in cell-cell communication and the maintenance of cellular homeostasis. They contain high levels of calcification inhibitors, such as fetuin-A and matrix Gla protein. Under pathological conditions (and particularly in the presence of uremic toxins), the secreted EVs acquire a pro-calcifying profile and thereby act as nucleating foci for the crystallization of hydroxyapatite and the propagation of calcification. Here, we review the most recent findings on the EVs’ pathophysiological role in VC, the impact of uremic toxins on EV biogenesis and functions, the use of EVs as diagnostic biomarkers and the EVs’ therapeutic potential in CKD.

Extracellular Vesicles as Potential Biomarkers for Early Detection and Diagnosis of Pancreatic Cancer.

Pancreatic carcinoma (PC) is highly metastatic, and it tends to be detected at advanced stages. Identifying and developing biomarkers for early detection of PC is crucial for a potentially curative treatment. Extracellular vesicles (EVs) are bilayer lipid membrane-structured nanovesicles found in various human bodily fluids, and they play important roles in tumor biogenesis and metastasis. Cancer-derived EVs are enriched with DNA, RNA, protein, and lipid, and they have emerged as attractive diagnostic biomarkers for early detection of PC. In this article, we provided an overview of the cell biology of EVs and their isolation and analysis, and their roles in cancer pathogenesis and progression. Multiplatform analyses of plasma-based exosomes for genomic DNA, micro RNA, mRNA, circular RNA, and protein for diagnosis of PC were critically reviewed. Numerous lines of evidence demonstrate that liquid biopsy with analysis of EV-based biomarkers has variable performance for diagnosis of PC. Future investigation is indicated to optimize the methodology for isolating and analyzing EVs and to identify the combination of EV-based biomarkers and other clinical datasets, with the goal of improving the predictive value, sensitivity, and specificity of screening tests for early detection and diagnosis of PC.

Uncovering temperature-dependent extracellular vesicle secretion in breast cancer.

In all living things, temperature is a key factor to maintain function and survive. Animals and plants need to adapt temperature change with optimizing their behaviour and growth by sensing temperature. Similarly, tumour cells must adapt continuously to fluctuations in external conditions including temperature. To find a better environment, cancer cells promote growth and metastasis, which contributes to tumour malignancy. Pathological studies in breast cancer have implied that temperature is associated with disease progression. However, no clear mechanisms have emerged for how thermal changes affect tumour cells and their gene regulation in tumour development and malignancy. Here we discovered the temperature‐dependent extracellular vesicle (EV) secretion in breast cancer. Cancer cell growth and EV secretion increased in a temperature‐dependent manner, which indicated that temperatures were associated with poor prognosis in breast cancer patients. We also found that low‐density lipoprotein receptor (LDLR), a responsible gene for temperature‐dependent EV secretion, was upregulated with the increase in temperature. Consistent with our results, LDLR gene has been characterized and identified as a key factor for malignancy in a wide range of cancers. Our findings shed new light on tumour aggressiveness and therapeutic strategies for breast cancer, especially regarding EV formation and secretion, thus providing a new relationship between cancer and EV biology in the light of temperature.

RNA packaging into extracellular vesicles: An orchestra of RNA-binding proteins?

Extracellular vesicles (EVs) are heterogeneous membranous particles released from the cells through different biogenetic and secretory mechanisms. We now conceive EVs as shuttles mediating cellular communication, carrying a variety of molecules resulting from intracellular homeostatic mechanisms. The RNA is a widely detected cargo and, impressively, a recognized functional intermediate that elects EVs as modulators of cancer cell phenotypes, determinants of disease spreading, cell surrogates in regenerative medicine, and a source for non‐invasive molecular diagnostics. The mechanistic elucidation of the intracellular events responsible for the engagement of RNA into EVs will significantly improve the comprehension and possibly the prediction of EV “quality” in association with cell physiology. Interestingly, the application of multidisciplinary approaches, including biochemical as well as cell‐based and computational strategies, is increasingly revealing an active RNA‐packaging process implicating RNA‐binding proteins (RBPs) in the sorting of coding and non‐coding RNAs. In this review, we provide a comprehensive view of RBPs recently emerging as part of the EV biology, considering the scenarios where: (i) individual RBPs were detected in EVs along with their RNA substrates, (ii) RBPs were detected in EVs with inferred RNA targets, and (iii) EV‐transcripts were found to harbour sequence motifs mirroring the activity of RBPs. Proteins so far identified are members of the hnRNP family (hnRNPA2B1, hnRNPC1, hnRNPG, hnRNPH1, hnRNPK, and hnRNPQ), as well as YBX1, HuR, AGO2, IGF2BP1, MEX3C, ANXA2, ALIX, NCL, FUS, TDP‐43, MVP, LIN28, SRP9/14, QKI, and TERT. We describe the RBPs based on protein domain features, current knowledge on the association with human diseases, recognition of RNA consensus motifs, and the need to clarify the functional significance in different cellular contexts. We also summarize data on previously identified RBP inhibitor small molecules that could also be introduced in EV research as potential modulators of vesicular RNA sorting.

Shedding Light on Extracellular Vesicle Biogenesis and Bioengineering.

Extracellular vesicles (EVs) are biocompatible, nano‐sized secreted vesicles containing many types of biomolecules, including proteins, RNAs, DNAs, lipids, and metabolites. Their low immunogenicity and ability to functionally modify recipient cells by transferring diverse bioactive constituents make them an excellent candidate for a next‐generation drug delivery system. Here, the recent advances in EV biology and emerging strategies of EV bioengineering are summarized, and the prospects for clinical translation of bioengineered EVs and the challenges to be overcome are discussed.

Preparation and characterization of extracellular vesicles.

Extracellular vesicles (EVs) are heterogeneous membranous vesicles secreted by every cell type and offer significant potential in therapy and diagnostics. Differential ultracentrifugation is the gold standard for EV isolation, although other techniques including, polyethylene glycol (PEG) precipitation, immunoprecipitation, size exclusion chromatography, and immuno-isolation approaches are common. Purified EVs can be characterized based on their physical characteristics, biochemical composition, or cell of origin. For size and concentration measurement, nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and electron microscopy are commonly employed methods. Biochemical analyses of EVs are typically performed using flow cytometry, immunoblotting, or proteomic investigation. Based on tissue of origin, EVs have specific markers that can be used to isolate and purify specific cell-associated EVs using an affinity selection approach. Despite existence of several methods for isolation and characterization, major limitations associated with each method hinder the progress of the field. Evolving concepts in EV biology possess great promise for better isolation and characterization leading to a better insight of biological function and have immense clinical implications. In this review, we discuss recent advancements in EV isolation and characterization approaches.

EVs as Potential New Therapeutic Tool/Target in Gastrointestinal Cancer and HCC.

Simple SummaryExtracellular vesicles (EVs)– cell derived organic, nano-sized particles that are more then only cell dust. Nowadays, EVs are a scientifically accepted novel route for cells, including tumour cells, to communicate and influence their host, orchestrating cellular processes from simple tumour growth to complex mechanism of tumour tolerance. Here we show the current understanding of those EVs in gastrointestinal cancer and hepatocellular carcinoma (HCC). The review emphasizes the potential of EVs as a new therapeutic tool and drug target. It critically evaluates their role, in an informative manner using cutting edge, current publications. Many of the cited work are regarded as milestones in the EV field of research drawing a promising perspective for future EV studies.For more than a decade, extracellular vesicles (EVs) have been in focus of science. Once thought to be an efficient way to eliminate undesirable cell content, EVs are now well-accepted as being an important alternative to cytokines and chemokines in cell-to-cell communication route. With their cargos, mainly consisting of functional proteins, lipids and nucleic acids, they can activate signalling cascades and thus change the phenotype of recipient cells at local and systemic levels. Their substantial role as modulators of various physiological and pathological processes is acknowledged. Importantly, more and more evidence arises that EVs play a pivotal role in many stages of carcinogenesis. Via EV-mediated communication, tumour cells can manipulate cells from host immune system or from the tumour microenvironment, and, ultimately, they promote tumour progression and modulate host immunity towards tumour’s favour. Additionally, the role of EVs in modulating resistance to pharmacological and radiological therapy of many cancer types has become evident lately. Our understanding of EV biology and their role in cancer promotion and drug resistance has evolved considerably in recent years. In this review, we specifically discuss the current knowledge on the association between EVs and gastrointestinal (GI) and liver cancers, including their potential for diagnosis and treatment.

Extracellular vesicles in spontaneous preterm birth.

Feto-maternal communication helps to maintain pregnancy and contributes to parturition at term and preterm. Endocrine and immune factor are well-reported communication mediators. Recent advances in extracellular vesicle (EV) biology have introduced them as major communication channels between the mother and fetus. EVs are round structures with a lipid bilayer membrane. EVs are generally categorized based on their size and mode of biogenesis. The most commonly reported EVs are exosomes with a size range of 30-160nm that are formed inside the intraluminal vesicles of multivesicular body. Microvesicles (MVs) are larger than>200nm and formed by outward budding of plasma membrane. Vesicles are released from all cells and carry various factors that reflect the physiologic state of cell at the time of their release. Analysis of vesicle provides a snapshot of origin cell. Recent studies in perinatal medicine have shown that exosomes are key communicators between feto-maternal units, and they can cross placenta. Fetal-derived exosomes released under term labor-associated conditions can cause parturition-associated changes in maternal uterine tissues. Exosomes carrying inflammatory cargo can cause preterm birth in animal models suggesting their functional role in parturition. A few reports have profiled differences between exosome cargos from term and preterm pregnancies and indicated their biomarker potential to predict high-risk pregnancy status. There are hardly any reports on MVs and their functional roles in reproduction. Herein, we review of EVs and MVs, their characteristics, function, and usefulness predicting adverse pregnancy complications such as preterm birth.

Extracellular vesicles: new players in regulating vascular barrier function.

Extracellular vesicles (EVs) have attracted rising interests in the cardiovascular field not only because they serve as serological markers for circulatory disorders but also because they participate in important physiological responses to stress and inflammation. In the circulation, these membranous vesicles are mainly derived from blood or vascular cells, and they carry cargos with distinct molecular signatures reflecting the origin and activation state of parent cells that produce them, thus providing a powerful tool for diagnosis and prognosis of pathological conditions. Functionally, circulating EVs mediate tissue-tissue communication by transporting bioactive cargos to local and distant sites, where they directly interact with target cells to alter their function. Recent evidence points to the critical contributions of EVs to the pathogenesis of vascular endothelial barrier dysfunction during inflammatory response to injury or infection. In this review, we provide a brief summary of the current knowledge on EV biology and advanced techniques in EV isolation and characterization. This is followed by a discussion focusing on the role and mechanisms of EVs in regulating blood-endothelium interactions and vascular permeability during inflammation. We conclude with a translational perspective on the diagnostic and therapeutic potential of EVs in vascular injury or infectious diseases, such as COVID-19.

Stoking the Fire: How Dying Cells Propagate Inflammatory Signalling through Extracellular Vesicle Trafficking.

Communication between dying cells and their environment is a critical process that promotes tissue homeostasis during normal cellular turnover, whilst during disease settings, it can contribute to inflammation through the release of intracellular factors. Extracellular vesicles (EVs) are a heterogeneous class of membrane-bound cell-derived structures that can engage in intercellular communication via the trafficking of bioactive molecules between cells and tissues. In addition to the well-described functions of EVs derived from living cells, the ability of dying cells to release EVs capable of mediating functions on target cells or tissues is also of significant interest. In particular, during inflammatory settings such as acute tissue injury, infection and autoimmunity, the EV-mediated transfer of proinflammatory cargo from dying cells is an important process that can elicit profound proinflammatory effects in recipient cells and tissues. Furthermore, the biogenesis of EVs via unique cell-death-associated pathways has also been recently described, highlighting an emerging niche in EV biology. This review outlines the mechanisms and functions of dying-cell-derived EVs and their ability to drive inflammation during various modes of cell death, whilst reflecting on the challenges and knowledge gaps in investigating this subgenre of extracellular vesicles research.

Paediatric/congenital cardiology physician scientists-An endangered species.

Producing excellent physician scientists starts with the active discovery of talent and dedication, supported by the strong belief that physician involvement in biomedical research is essential to make fundamental discoveries that improve human health. The revolution of surgical and interventional therapy of structural heart disease has had 'profoundly positive effects on survival and quality of life over the decades. (…) Small increments in clinical improvement will still be possible in the future, but for the most part, the potential for major advancement using these techniques has been exhausted' (Frank Hanley, MD; Stanford). Personalized medicine, rapid genetic diagnostics, RNA and extracellular vesicle biology, epigenetics, gene editing, gene and stem cell-derived therapy are exemplary areas where specialized training for paediatric/congenital cardiology physician scientists will be increasingly needed to further advance the field. About a decade ago, a series in Circulation discussed academic career models and highlighted the major challenges facing the cardiovascular 'clinician scientist' (syn. physician scientist), which have not abated since. To develop the skills and expertise in both clinical congenital cardiology and basic research, the training of fellows must be focused and integrated. The current pandemic COVID-19 puts additional pressure and hurdles on fellows-in-training (FIT) and early career investigators (ECI) who aim to establish, consolidate or expand their own research group. Here, we discuss the major challenges, opportunities and necessary changes for academic institutions to sustain and recruit physician scientists in paediatric/congenital cardiology in the years to come.

Emerging roles of extracellular vesicles in the intercellular communication for exercise-induced adaptations.

Complex organisms rely heavily on intercellular communication. The rapidly expanding field of extracellular vesicle biology has made it clear that the necessary intercellular communication occurs partly through their paracrine and endocrine actions. Extracellular vesicles are nanoscale lipid membranes (30-2,000 nm in diameter) that shuttle functional biological material between cells. They are released from numerous tissues and are isolated from nearly all biofluids and cell cultures. Although their biogenesis, cell targeting, and functional roles are incompletely understood, they appear to have crucial roles in physiological and disease processes. Their enormous potential to serve as sensitive biomarkers of disease and also new therapeutic interventions for diseases have gained them considerable attention in recent years. Regular physical exercise training confers systemic health benefits and consequently prevents many age-related degenerative diseases. Many of the molecular mechanisms responsible for the salubrious effects of exercise are known, yet a common underlying mechanism potentially responsible for the holistic health benefits of exercise has only recently been explored (i.e., via extracellular vesicle transport of biological material). Here, we provide an overview of extracellular vesicle biology before outlining the current evidence on the capacity for a single bout and chronic exercise to elicit changes in extracellular vesicle content and modulate their molecular cargo (e.g., small RNAs). We highlight areas for future research and emphasize their potential utility as biomarkers and therapeutic strategies of disease and its prevention.