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The catalytic action of invertase generates bilayer asymmetry that stabilises membrane curvature. The driving mechanism for the generation of membrane curvature by invertase is investigated using giant unilamellar vesicles (GUVs). The invertase cleaves the sucrose in the exterior compartment, thereby creating a sugar asymmetry across the bilayer membrane that is measured for GUV membranes consisting of the lipid Dioleoylphosphatidylcholine (DOPC). Finally, the advantage of this method to control membrane curvature and to stabilize multi‐sphere morphologies is demonstrated. The GUV system in the presence of invertase is beneficial as a tool to generate multiple on‐demand compartments with more extended stability after the enzymatic activity has established the asymmetry. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is the most frequent malignancy among primary brain tumors in adults and one of the worst 5-year survival rates (< 7%) among all human cancers. Till now, treatments that target particular cell or intracellular metabolism have not improved patients' survival. GBM recruits healthy brain cells and subverts their processes to create a microenvironment that contributes to supporting tumor progression. This microenvironment encompasses a complex network in which malignant cells interact with each other and with normal and immune cells to promote tumor proliferation, angiogenesis, metastasis, immune suppression, and treatment resistance. Communication can be direct via cell-to-cell contact, mainly through adhesion molecules, tunneling nanotubes, gap junctions, or indirect by conventional paracrine signaling by cytokine, neurotransmitter, and extracellular vesicles. Understanding these communication routes could open up new avenues for the treatment of this lethal tumor. Hence, therapeutic approaches based on glioma cells' communication have recently drawn attention. This review summarizes recent findings on the crosstalk between glioblastoma cells and their tumor microenvironment, and the impact of this conversation on glioblastoma progression. We also discuss the mechanism of communication of glioma cells and their importance as therapeutic targets and diagnostic and prognostic biomarkers. Overall, understanding the biological mechanism of specific interactions in the tumor microenvironment may help in predicting patient prognosis and developing novel therapeutic strategies to target GBM. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the third most common cancer and the leading cause of cancer death globally. Resistance to therapy is a challenge for CRC treatment. Mesenchymal stem cells (MSCs) have become one of the furthermost effective approaches for tumor treatment due to their specific feature; however, their therapeutic function is controversial. Recently, extracellular vesicles (EVs) derived from MSCs (MSCs-EVs) have attracted extensive research attention due to their promising role in CRC treatment. EVs are cell-derived vesicles that transfer different biomolecules between cells, contributing to intracellular communication. MSCs-EVs can suppress CRC by delivering therapeutic agents to tumor cells. Several studies indicate that MSCs-EVs can serve as a drug delivery system for the treatment of different cancers. Various methods are used to modify (engineer) MSCs-EVs for loading therapeutic agents. Modified MSCs-EVs have improved specificity, targeting ability, and immunogenicity compared to synthetic carriers. Furthermore, CRC-EVs participate in regulating different cells, such as immune cells, fibroblasts, and endothelial cells, promoting tumorigenesis. MSCs-EVs-based therapy indicates a high potential in the treatment of cancer; however, the majority of studies have been conducted in the pre-clinical, and their clinical applications need further scrutiny. In this review, we describe the biogenesis of EVs, focusing on the effect of MSCs-EVs on CRC cells and CRC-derived EVs on other cells. Furthermore, MSCs-EVs as a drug delivery system for cancers is also reviewed, and perspectives regarding the therapeutic application of MSCs-EVs are discussed. [ABSTRACT FROM AUTHOR]

The rapid development of aberrant cells outgrowing their normal bounds, which can subsequently infect other body parts and spread to other organs—a process known as metastasis—is one of the significant characteristics of cancer. The main reason why cancer patients die is because of widespread metastases. This abnormal cell proliferation varies in cancers of over a hundred types, and their response to treatment can vary substantially. Several anti-cancer drugs have been discovered to treat various tumors, yet they still have harmful side-effects. Finding novel, highly efficient targeted therapies based on modifications in the molecular biology of tumor cells is essential to reduce the indiscriminate destruction of healthy cells. Exosomes, an extracellular vesicle, are promising as a drug carrier for cancer therapy due to their good tolerance in the body. In addition, the tumor microenvironment is a potential target to regulate in cancer treatment. Therefore, macrophages are polarized toward M1 and M2 phenotypes, which are involved in cancer proliferation and are malignant. It is evident from recent studies that controlled macrophage polarization might contribute to cancer treatment, by the direct way of using miRNA. This review provides an insight into the potential use of exosomes to develop an 'indirect', more natural, and harmless cancer treatment through regulating macrophage polarization. [ABSTRACT FROM AUTHOR]

The interaction of many peptides with lipid bilayer membranes strongly depends on the lipid composition. Here, a study of the impact of unsaturated lipid acyl chains on the interaction of a derivative of the HIV-1 fusion peptide with lipid bilayer vesicles is presented. Lipid bilayer vesicles composed of mixtures of lipids with two saturated acyl chains and lipids and one saturated and one unsaturated acyl chain, but identical head groups, were studied. The dependence of the peptide conformation on the unsaturated lipid content was probed by circular dichroism spectroscopy, while the impact of the peptide on the bilayer structure was determined by small-angle neutron scattering. The impact of the peptide on the lipid bilayer vesicle dynamics was investigated using neutron spin echo spectroscopy. Molecular dynamics simulations were used to characterize the behavior of the systems studied to determine if there were clear differences in their physical properties. The results reveal that the peptide–bilayer interaction is not a simple function of the unsaturated lipid acyl chain content of the bilayer. Instead, the peptide behavior is more consistent with that seen for the bilayer containing only unsaturated lipids, which is supported by lipid-specific interactions revealed by the simulations. [ABSTRACT FROM AUTHOR]

Acute myocardial infarction (AMI) is a severe ischemic disease with high morbidity and mortality worldwide. Maladaptive cardiac remodeling is a series of abnormalities in cardiac structure and function that occurs following myocardial infarction (MI). The pathophysiology of this process can be separated into two distinct phases: the initial inflammatory response, and the subsequent longer-term scar revision that includes the regression of inflammation, neovascularization, and fibrotic scar formation. Extracellular vesicles are nano-sized lipid bilayer vesicles released into the extracellular environment by eukaryotic cells, containing bioinformatic transmitters which are essential mediators of intercellular communication. EVs of different cellular origins play an essential role in cardiac remodeling after myocardial infarction. In this review, we first introduce the pathophysiology of post-infarction cardiac remodeling, as well as the biogenesis, classification, delivery, and functions of EVs. Then, we explore the dual role of these small molecule transmitters delivered by EVs in post-infarction cardiac remodeling, including the double-edged sword of pro-and anti-inflammation, and pro-and anti-fibrosis, which is significant for post-infarction cardiac repair. Finally, we discuss the pharmacological and engineered targeting of EVs for promoting heart repair after MI, thus revealing the potential value of targeted modulation of EVs and its use as a drug delivery vehicle in the therapeutic process of post-infarction cardiac remodeling. [ABSTRACT FROM AUTHOR]

Tendon mimetic scaffolds that recreate the tendon hierarchical structure and niche have increasing potential to fully restore tendon functionality. However, most scaffolds lack biofunctionality to boost the tenogenic differentiation of stem cells. In this study, we assessed the role of platelet-derived extracellular vesicles (EVs) in stem cells' tenogenic commitment using a 3D bioengineered in vitro tendon model. First, we relied on fibrous scaffolds coated with collagen hydrogels encapsulating human adipose-derived stem cells (hASCs) to bioengineer our composite living fibers. We found that the hASCs in our fibers showed high elongation and cytoskeleton anisotropic organization, typical of tenocytes. Moreover, acting as biological cues, platelet-derived EVs boosted the hASCs' tenogenic commitment, prevented phenotypic drift, enhanced the deposition of the tendon-like extracellular matrix, and induced lower collagen matrix contraction. In conclusion, our living fibers provided an in vitro system for tendon tissue engineering, allowing us to study not only the tendon microenvironment but also the influence of biochemical cues on stem cell behavior. More importantly, we showed that platelet-derived EVs are a promising biochemical tool for tissue engineering and regenerative medicine applications that are worthy of further exploration, as paracrine signaling might potentiate tendon repair and regeneration. [ABSTRACT FROM AUTHOR]

Background: Extracellular vesicles (EVs), including small EVs (sEVs) such as exosomes, exhibit great potential for the diagnosis and treatment of brain disorders, representing a valuable tool for precision medicine. The latter demands high-quality human biospecimens, especially in complex disorders in which pathological and specimen heterogeneity, as well as diverse individual clinical profile, often complicate the development of precision therapeutic schemes and patient-tailored treatments. Thus, the collection and characterization of physiologically relevant sEVs are of the utmost importance. However, standard brain EV isolation approaches rely on tissue dissociation, which can contaminate EV fractions with intracellular vesicles. Methods: Based on multiscale analytical platforms such as cryo-EM, label-free proteomics, advanced flow cytometry, and ExoView analyses, we compared and characterized the EV fraction isolated with this novel method with a classical digestion-based EV isolation procedure. Moreover, EV biogenesis was pharmacologically manipulated with either GW4869 or picrotoxin to assess the validity of the spontaneous-release method, while the injection of labelled-EVs into the mouse brain further supported the integrity of the isolated vesicles. Results: We hereby present an efficient purification method that captures a sEV-enriched population spontaneously released by mouse and human brain tissue. In addition, we tested the significance of the release method under conditions where biogenesis/secretion of sEVs was pharmacologically manipulated, as well as under animals' exposure to chronic stress, a clinically relevant precipitant of brain pathologies, such as depression and Alzheimer's disease. Our findings show that the released method monitors the drug-evoked inhibition or enhancement of sEVs secretion while chronic stress induces the secretion of brain exosomes accompanied by memory loss and mood deficits suggesting a potential role of sEVs in the brain response to stress and related stress-driven brain pathology. Conclusions: Overall, the spontaneous release method of sEV yield may contribute to the characterization and biomarker profile of physiologically relevant brain-derived sEVs in brain function and pathology. 7SKHktGsByupRjRZYNwNfM Video Abstract [ABSTRACT FROM AUTHOR]

Lymph node metastasis is related to poor prognosis in oral squamous cell carcinoma (OSCC), and few studies have explored the relevance of postoperative drainage fluid (PDF) in metastasis. Extracellular vesicles (EVs) are nanosized vesicles that can transfer oncogenic molecules to regulate tumorigenesis. However, the proteomic profile of postoperative drainage fluid–derived EVs (PDF-EVs) in OSCC has not been elucidated. Herein, we collected drainage fluid from OSCC patients after neck dissection to investigate the difference in PDF-EVs between patients with metastatic lymph nodes (the LN+ group) and nonmetastatic lymph nodes (the LN− group). The proteomic profile of PDF-EVs from the LN+ and LN− groups was compared using label-free liquid chromatography tandem-mass spectrometry–based protein quantification. The results revealed that PDF-EVs were mainly derived from epithelial cells and immune cells. A total of 2,134 proteins in the PDF-EVs were identified, and 313 were differentially expressed between the LN+ and LN− groups. Metabolic proteins, such as EHD2 and CAVIN1, were expressed at higher levels in the LN+ group than in the LN− group, and the levels of EHD2 and CAVIN1 in the postoperative drainage fluid were positively correlated with lymph node metastasis. Our study revealed previously undocumented postoperative drainage fluid–associated proteins in patients with metastatic OSCC, providing a starting point for understanding their role in metastatic and nonmetastatic OSCC. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EV) are a novel biomarker source for diagnosis and prognosis of cardiovascular disease. A protein comparison of plasma EVs in relation to blood plasma and atherosclerotic plaque has not been performed but would provide insight into the origin and content of biomarker sources and their association with atherosclerotic progression. Using samples of 88 carotid endarterectomy patients in the Athero-Express, 92 proteins (Olink Cardiovascular III panel) were measured in citrate plasma, plasma derived LDL-EVs and atherosclerotic plaque. Proteins were correlated between sources and were related to pre-operative stroke and 3-year major adverse cardiovascular events (MACE). Plasma and EV proteins correlated moderately on average, but with substantial variability. Both showed little correlation with plaque, suggesting that these circulating biomarkers may not originate from the latter. Plaque (n = 17) contained most differentially-expressed proteins in patients with stroke, opposed to EVs (n = 6) and plasma (n = 5). In contrast, EVs contained most differentially-expressed proteins for MACE (n = 21) compared to plasma (n = 9) and plaque (n = 1). EVs appear to provide additional information about severity and progression of systemic atherosclerosis than can be obtained from plasma or atherosclerotic plaque. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are a unique mode of intercellular communication capable of specificity in transmitting signals and cargo to coordinate local and distant cellular functions. A key example of this is the essential role that EVs secreted by epithelial cells lining the lumen of the male reproductive tract play in post-spermatogenic sperm maturation. We recently showed in a preclinical mouse model that this fundamental process had a causal role in somatic-to-germline transmission of biological information regarding prior stress experience capable of altering the rate of fetal development. However, critical mechanistic questions remain unanswered as to the processes by which signaling occurs between EVs and sperm, and whether EVs or their cargo are delivered at conception and are detectable in the early embryo. Unfortunately, notable methodological limitations shared across EV biology, particularly in the isolation and labeling of EVs, complicate efforts to answer these important questions as well as questions on EV targeting specificity and mechanisms. In our current studies, we developed a novel approach to track EVs using a conditional transgenic construct designed to label EVs via conditional Cre-induced hemagglutinin (HA) tagging of the EV endogenous tetraspanin, CD63. In our exhaustive validation steps, this internal small molecular weight tag did not affect EV secretion or functionality, a common problem found in the previous design of EV tags using larger molecular weight proteins, including fluorescent proteins. Utilizing a stably transfected immortalized epididymal epithelial cell line, we first validated key parameters of the conditional HA-tagged protein packaged into secreted EVs. Importantly, we systematically confirmed that expression of the CD63-HA had no impact on the production, size distribution, or surface charge of secreted EVs, nor did it alter the tetraspanin or miRNA composition of these EVs. We also utilized the CD63-HA EVs to verify physical interactions with sperm. Finally, using in vitro fertilization we produced some of the first images confirming sperm delivered EV cargo at conception and still detectable in the early-stage embryo. As such, this construct serves as a methodological advance and as a valuable tool, with applications in the study of EV function across biomedical research areas. [ABSTRACT FROM AUTHOR]

Exosomes are nano-sized extracellular vesicles that regulate cell growth and defense by delivering bioactive cellular constituents. They are a promising material for biomedical and cosmetic utilization, especially in medicinal crops such as ginseng. One main hurdle to their usage is the need for a method to isolate stable exosomes with high purity. In this study, we first tested two methods to isolate exosomes from ginseng: ultracentrifugation, the most widely used method; and the ExoQuick system, a polymer-based exosome precipitation approach. We also designed and tested a third method in which we combined ultracentrifugation and ExoQuick methods. Size distribution analysis revealed that the exosome isolation purity by the ultracentrifugation and ExoQuick methods alone were 34.1% and 59.7%, respectively, while the combination method greatly improved exosome isolation purity (83.3%). Furthermore, we found that the combination method also increases the colloidal stability of isolated ginseng exosomes, and the increase was almost double that of the ultracentrifugation method. Lastly, we showed that the combination method can also be used to isolate high-purity and high-stability exosomes from the model plant Arabidopsis. Overall, our findings indicate that the combination method is suitable to isolate high-purity and high-stability exosomes from plants including ginseng. [ABSTRACT FROM AUTHOR]

Background: Exosomes are nanosized membranous vesicles secreted by various types of cells, which facilitate intercellular communication by transporting bioactive compounds. Exosomes are abundant in biological fluids including semen, and their protein composition and the potential of seminal plasma exosomes (SPEs) as fertility biomarkers were elucidated in humans, however, little information is available regarding buffalo (Bubalus bubalis). Here, we examined protein correlation between spermatozoa, seminal plasma (SP), and SPEs, and we compared and analyzed protein differences between high-motility (H-motility) and low-motility (L-motility) SPEs in buffalo. Results: SPEs were concentrated and purified by ultracentrifugation combined with sucrose density gradient centrifugation, followed by verification using western blotting, nanoparticle tracking analysis, and transmission electron microscopy. Protein composition in spermatozoa, SP and SPEs, and protein difference in H- and L-motility SPEs were identified by LC-MS/MS proteomic analysis and were functionally analyzed through comprehensive bioinformatics. Many SPEs proteins originated from spermatozoa and SP, and nearly one third were also present in spermatozoa and SP. A series of proteins associated with reproductive processes including sperm capacitation, spermatid differentiation, fertilization, sperm-egg recognition, membrane fusion, and acrosome reaction were integrated in a functional network. Comparative proteomic analyses showed 119 down-regulated and 41 up-regulated proteins in L-motility SPEs, compared with H-motility SPEs. Gene Ontology (GO) enrichment of differentially expressed proteins (DEPs) showed that most differential proteins were located in sperm and vesicles, with activities of hydrolase and metalloproteinase, and were involved in sperm-egg recognition, fertilization, single fertilization, and sperm-zona pellucida binding processes, etc. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that differential proteins were mainly involved in the PPRP signaling pathway, calcium signaling pathway, and cAMP signaling pathway, among others. Furthermore, 6 proteins associated with reproduction were validated by parallel reaction monitoring analysis. Conclusion: This study provides a comprehensive description of the seminal plasma exosome proteome and may be of use for further screening of biomarkers associated with male infertility. [ABSTRACT FROM AUTHOR]

Exosomes are small vesicles that contain diverse miRNA, mRNA, and proteins that are secreted by multiple cells, and play a vital function in cell-- cell communication. Numerous exosomes produced by cells have been demonstrated to be protective against spinal cord injury (SCI). This study aims to investigate the neuroprotective effect of Schwann cells-derived exosomes (SCs-Exos) on spinal cord injury. We found that SCs-Exos can be taken directly by brain-derived endothelial cells.3 (bEnd.3 cells) and promoted to proliferate, migrate, and form bEnd.3 tube. Additionally, our results showed that the pro-angiogenesis molecules, Integrin-b1, were highly expressed in SCs-Exos. Moreover, we used special shRNA technology to investigate the role of Integrin-b1 in mediating the effect of SCs-Exos-induced angiogenesis on bEnd.3 cells. We observed that the pro-angiogenic effect of SCs-Exos on bEnd.3 cells was suppressed by inhibiting the expression of integrin-b1 in SCs-Exos. In the SCI model, we found that SCs-Exos attenuated tissue damage and improved functional recovery after SCI. Using immunofluorescence staining, we observed that SCs-Exos treatment promoted angiogenesis in SCI, and integrin-b1 was required to promote angiogenesis. In conclusion, our results indicate that SCs-Exos promote angiogenesis by delivering integrin-b1 and may serve as a promising novel therapeutic agent for enhancing neurological functional recovery after SCI. [ABSTRACT FROM AUTHOR]

Hypertension may develop before or after the onset of diabetes and it is known to increase the risk of developing diabetic nephropathy. Alpha-1 antitrypsin (AAT) is a multi-functional protein with beneficial effects in various diseases but its role in reducing blood pressure in the diabetic kidney has not been thoroughly studied. Like blood pressure, epithelial sodium channels (ENaC) and its adaptor protein myristoylated alanine-rich C-kinase substrate (MARCKS) are regulated by circadian rhythms. Our hypothesis is that administration of human AAT (hAAT) reduces blood pressure in hypertensive diabetic mice by attenuating membrane expression of ENaC and its association with the actin cytoskeleton. First, we show hAAT administration results in reduced blood pressure in diabetic db/db mice compared to vehicle treatment in both the inactive and active cycles. Western blotting and immunohistochemistry analyses showed a reduction of ENaC and the actin cytoskeleton protein, MARCKS in the kidneys of diabetic db/db mice treated with hAAT compared to vehicle. hAAT treatment resulted in elevated amounts of extracellular vesicles present in the urine of diabetic db/db mice compared to vehicle treatment both in the inactive and active cycles. Multiple hexosylceramides, among other lipid classes increased in urinary EVs released from hAAT treated hypertensive diabetic mice compared to vehicle treated mice. Taken together, these data suggest hAAT treatment could normalize blood pressure in the diabetic kidney in a mechanism involving attenuation of renal ENaC and MARCKS protein expression and possibly ceramide metabolism to hexosylceramide in kidney cells. [ABSTRACT FROM AUTHOR]

Background: Most pediatric patients show a decline in platelet counts while on extracorporeal life support (ECLS) and require multiple platelet transfusions. To better understand platelet loss during ECLS, this study estimated platelet loss rates due to diagnostic phlebotomy, platelet activation, bleeding and other causes. Methods: We collected data on 91 patients (1d‐20y, 50 M, 41F). Platelet losses were estimated based on changes in platelet count, patient+circuit blood volume, and transfused platelet volumes. Platelet extracellular vesicles were measured by flow cytometry. Results: Median platelet loss was 2.8 × 109/L/hr, more than twice the normal rate of platelet removal and equivalent to a 67 000/μl decrease in platelet count per day. While platelet loss was correlated with platelet transfusion (r2 = 0.51), transfusion underestimated platelet loss in patients with large decreases in platelet count and over‐estimated platelet loss in neonates where the circuit volume > patient blood volume. Patients with disseminated intravascular coagulation before or significant bleeding during ECLS have double the rate of platelet loss. Platelet activation accounted for ~32% of total platelet loss, bleeding ~36% and phlebotomy 4%, with the remaining one‐third due to other causes. Annexin‐negative platelet extracellular vesicle release, a measure of platelet damage, was increased 9‐fold during ECLS. Conclusion: Our study is the first to quantitate total, phlebotomy and activation related platelet loss during ECLS. Platelet activation accounts for ~32% of total platelet loss, while bleeding doubles the platelet loss rate. The etiology of the remaining platelet loss is unknown. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are potent means of cell-to-cell communication. They are released in biological fluids, including blood, urine, and saliva, and can be exploited to identify new biomarkers of diseases. We hypothesized that EVs contain molecular cargos involved in bone metabolism, possibly mirroring biological differences between postmenopausal and disuse osteoporosis. We tested this hypothesis in primary murine osteoblasts subjected to steroid depletion or to unloading, and in the serum of animal models of osteoporosis induced by ovariectomy or hindlimb tail suspension. EVs were isolated by ultracentrifugation and analysed by transmission electron microscopy, cytofluorimetry, immunoblotting and RT-PCR. Large-scale analyses were performed by Real-Time arrays and Proteome Profiler™ Antibody arrays. Finally, precise titration of analytes was carried out by ELISA assay. In vitro, we confirmed an increased release of EVs enriched in surface RANKL by primary mouse osteoblasts subjected to steroid depletion or simulated microgravity compared to controls. In vivo, circulating EVs isolated from the sera of control female mice expressed RANKL along with other genes associated with bone metabolism. Serum EVs from ovariectomized or hindlimb tail-suspended mice showed distinct molecular profiles. They expressed RANKL with different kinetics, while transcriptomic and proteomic profiles uncovered unique molecular signatures that discriminated the two conditions, unveiling exclusive molecules expressed in time- and osteoporosis type-dependent manner. These results suggest that circulating EVs could represent a new tool for monitoring the onset and the progression of diverse types of the disease in mice, paving the way for their exploitation to diagnose human osteoporosis in liquid biopsies. [ABSTRACT FROM AUTHOR]

Extracellular vesicle (EV) research is a rapidly developing field, mainly due to the key role of EVs in intercellular communication and pathophysiological processes. However, the heterogeneity of EVs challenges their exploration and the establishment of gold-standard methods. Here, we aimed to reveal the influence of technical changes on EV biology and the reliability of experimental data. We used B16F1 melanoma cells as a model and applied nanoparticle tracking analysis, mass spectrometry (LC-MS/MS) and pathway enrichment analysis to analyze the quantity, size distribution, proteome and function of their small EVs (sEVs) produced in sEV-depleted fetal bovine serum (FBS)-containing medium or serum-free medium. Additionally, we investigated the effects of minor technical variances on the quality of sEV preparations. We found that storage of the isolates at −80 °C has no adverse effect on LC-MS/MS analysis, and an additional washing step after differential ultracentrifugation has a minor influence on the sEV proteome. In contrast, FBS starvation affects the production and proteome of sEVs; moreover, these vesicles may have a greater impact on protein metabolism, but a smaller impact on cell adhesion and membrane raft assembly, than the control sEVs. As we demonstrated that FBS starvation has a strong influence on sEV biology, applying serum-free conditions might be considered in in vitro sEV studies. [ABSTRACT FROM AUTHOR]

Neurodegenerative diseases constitute a group of pathologies whose etiology remains unknown in many cases, and there are no treatments that stop the progression of such diseases. Moreover, the existence of the blood–brain barrier is an impediment to the penetration of exogenous molecules, including those found in many drugs. Exosomes are extracellular vesicles secreted by a wide variety of cells, and their primary functions include intercellular communication, immune responses, human reproduction, and synaptic plasticity. Due to their natural origin and molecular similarities with most cell types, exosomes have emerged as promising therapeutic tools for numerous diseases. Specifically, neurodegenerative diseases have shown to be a potential target for this nanomedicine strategy due to the difficult access to the brain and the strategy's pathophysiological complexity. In this regard, this review explores the most important biological-origin drug delivery systems, innovative isolation methods of exosomes, their physicochemical characterization, drug loading, cutting-edge functionalization strategies to target them within the brain, the latest research studies in neurodegenerative diseases, and the future challenges of exosomes as nanomedicine-based therapeutic tools. [ABSTRACT FROM AUTHOR]

The management and screening of prostate cancer (PC) is still the main problem in clinical practice. In this study, we investigated the role of aggressiveness genetic markers for PC stratification. We analyzed 201 plasma samples from PC patients and controls by digital PCR. For selection and validation, 26 formalin-fixed paraffin-embedded tissues, 12 fresh tissues, and 24 plasma samples were characterized by RNA-Seq, immunochemistry, immunofluorescence, Western blot, and extracellular-vesicles analyses. We identified three novel non-invasive biomarkers; all with an increased expression pattern in patients (PCA3: p = 0.002, S100A4: p ≤ 0.0001 and MRC2: p = 0.005). S100A4 presents the most informative AUC (area under the curve) (0.735). Combination of S100A4, MRC2, and PCA3 increases the discriminatory power between patients and controls and between different more and less aggressive stages (AUC = 0.761, p ≤ 0.0001). However, although a sensitivity of 97.47% in PCA3 and a specificity of 90.32% in S100A4 was reached, the detection signal level could be variable in some analyses owing to tumor heterogeneity. This is the first time that the role of S100A4 and MRC2 has been described in PC aggressiveness. Moreover, the combination of S100A4, MRC2, and PCA3 has never been described as a non-invasive biomarker for PC screening and aggressiveness. [ABSTRACT FROM AUTHOR]

Atherosclerosis (AS) has emerged as one of the prevalent arterial vascular diseases characterized by plaque and inflammation, primarily causing disability and mortality globally. Drug therapy remains the main treatment for AS. However, a series of obstacles hinder effective drug delivery. Nature, from natural micro-/nano-structural biological particles like natural cells and extracellular vesicles to the distinctions between the normal and pathological microenvironment, offers compelling solutions for efficient drug delivery. Nature-inspired nanocarriers of synthetic stimulus-responsive materials and natural components, such as lipids, proteins and membrane structures, have emerged as promising candidates for fulfilling drug delivery needs. These nanocarriers offer several advantages, including prolonged blood circulation, targeted plaque delivery, targeted specific cells delivery and controlled drug release at the action site. In this review, we discuss the nature-inspired nanocarriers which leverage the natural properties of cells or the microenvironment to improve atherosclerotic drug therapy. Finally, we provide an overview of the challenges and opportunities of applying these innovative nature-inspired nanocarriers. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cells (MSCs) are a promising cell therapy due to their immunomodulatory function, which is mediated by extracellular vesicles (EVs). However, EV clinical translation has been unsuccessful due in part to EV functional heterogeneity. Priming MSCs with inflammatory signals can enhance immunomodulatory function, but priming approaches have not been investigated to increase the efficiency of EV manufacturing and reduce heterogeneity. MSCs expanded at a clinical scale are typically discarded after being primed and producing a single EV batch such that every batch incurs the full expansion cost. Thus, development of an approach to generate multiple batches of EVs from a single production of primed MSCs (termed "cyclical priming") could address these challenges. Adipose-derived MSCs were expanded in serum-containing (MSC-GM) or chemically defined (CDM) media and primed with 50 ng/mL interferon-gamma (IFN-γ)/tumor necrosis factor-alpha (TNF-α). The effects of priming and recovery were screened using a modified Cell Painting assay (nuclei, actin, endoplasmic reticulum, mitochondria stains) followed by high content imaging and multivariate morphological analysis. Indoleamine 2,3-dioxgenase (IDO) activity served as a functional screen across priming cycles and acridine orange/propidium iodide (AO/PI) staining was used to quantify viability. We have shown MSC morphological response predicts function and 50 ng/mL IFN-γ/TNF-α priming increases EV yield and potency in a bioreactor. Morphological profiling distinguished primed and recovered groups, which became larger and more elongated (Fig 1). Cyclical priming sustained higher IDO activity and viability compared to traditional priming (primed cycle 1) through 144 hours in culture (Figs 2, 3). CDM groups maintained similar IDO activity and higher live count and viability in late culture (Fig 3). This is the first demonstration of cyclical priming, which could be implemented in EV manufacturing. Furthermore, screening cyclical priming informs future larger scale experiments involving production of EVs in bioreactors and omics characterization and functional evaluation of EVs from multiple priming cycles towards understanding mechanisms of priming. This work advances methods to increase EV production from a single batch of cells, minimizing process cost and reducing batch-batch heterogeneity of cell products, which facilitates clinical translation of MSC-based therapies. [ABSTRACT FROM AUTHOR]

This correction notice addresses an error in the original article titled "Extracellular vesicles-coupled miRNAs from oviduct and uterus modulate signaling pathways related to lipid metabolism and bovine early embryo development." The authors reported that the last two sentences of the Results section in the Abstract were mistakenly placed at the end and should have been placed at the beginning. The corrected Results section provides information on the miRNA cargo in oviduct and uterus extracellular vesicles (OF-EVs and UF-EVs) during different phases of the luteal phase. It discusses the differentially expressed miRNAs and their predicted modulation of various pathways related to lipid metabolism and degradation. The study suggests that the differences in miRNAs between OF-EVs and UF-EVs may reflect different environments to meet the changing needs of the embryo, and miRNAs may play a role in regulating embryo lipid metabolism, immune system, and implantation. [Extracted from the article]

Abdominal aortic aneurysm (AAA) is a frequent aortic disease. If the diameter of the aorta is larger than 5 cm, an open surgical repair (OSR) or an endovascular aortic repair (EVAR) are recommended. To prevent possible complications (i.e., endoleaks), EVAR-treated patients need to be monitored for 5 years following the intervention, using computed tomography angiography (CTA). However, this radiological method involves high radiation exposure in terms of CTA/year. In such a context, the study of peripheral-blood-circulating extracellular vesicles (pbcEVs) has great potential to identify biomarkers for EVAR complications. We analyzed several phenotypes of pbcEVs using polychromatic flow cytometry in 22 patients with AAA eligible for EVAR. From each enrolled patient, peripheral blood samples were collected at AAA diagnosis, and after 1, 6, and 12 months following EVAR implantation, i.e. during the diagnostic follow-up protocol. Patients developing an endoleak displayed a significant decrease in activated-platelet-derived EVs between the baseline condition and 6 months after EVAR intervention. Furthermore, we also observed, that 1 month after EVAR implantation, patients developing an endoleak showed higher concentrations of activated-endothelial-derived EVs than patients who did not develop one, suggesting their great potential as a noninvasive and specific biomarker for early identification of EVAR complications. [ABSTRACT FROM AUTHOR]

Exosomes are endosome-derived extracellular vesicles involved in intercellular communication. They are generated as intraluminal vesicles within endosomal compartments that fuse with the plasma membrane (PM). The molecular events that generate secretory endosomes and lead to the release of exosomes are not well understood. We identified a subclass of nonproteolytic endosomes at prelysosomal stage as the compartment of origin of CD63 positive exosomes. These compartments undergo a Rab7a/Arl8b/Rab27a GTPase cascade to fuse with the PM. Dynamic endoplasmic reticulum (ER)-late endosome (LE) membrane contact sites (MCS) through ORP1L have the distinct capacity to modulate this process by affecting LE motility, maturation state, and small GTPase association. Thus, exosome secretion is a multi-step process regulated by GTPase switching and MCS, highlighting the ER as a new player in exosome-mediated intercellular communication. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) derived from human mesenchymal stem cells (hMSCs) have been widely known to have therapeutic effects by representing characteristics of the origin cells as an alternative for cell-based therapeutics. Major limitations of EVs for clinical applications include low production yields, unknown effects from serum impurities, and relatively low bioactivities against dose. In this study, we proposed a cell modulation method with melatonin for human umbilical cord MSCs (hUCMSCs) cultured in serum-free chemically defined media (CDM) to eliminate the effects of serum-derived impurities and promote regeneration-related activities. miRNAs highly associated with regeneration were selected and the expression levels of them were comparatively analyzed among various types of EVs depending on culture conditions. The EVs derived from melatonin-stimulated hUCMSCs in CDM (CDM mEVs) showed the highest expression levels of regeneration-related miRNAs, and 7 times more hsa-let-7b-5p, 5.6 times more hsa-miR-23a-3p, and 5.7 times more hsa-miR-100-5p than others, respectively. In addition, the upregulation of various functionalities, such as wound healing, angiogenesis, anti-inflammation, ROS scavenging, and anti-apoptosis, were proven using in vitro assays by simulating the characteristics of EVs with bioinformatics analysis. The present results suggest that the highly regenerative properties of hUCMSC-derived EVs were accomplished with melatonin stimulation in CDM and provided the potential for clinical uses of EVs. [ABSTRACT FROM AUTHOR]

Exosomes are nanosized extracellular vesicles secreted by various cell types, including those of the immune system, such as natural killer (NK) cells. They play a role in intercellular communication by transporting signal molecules between the cells. Recent studies have reported that NK cell-derived exosomes (NK-exo) contain cytotoxic proteins-induced cell death. However, the characteristics and potential functions of NK-exo, especially for the liver cancer are poorly understood. In this study, we investigated the anti-tumor effects of NK-exo in the primary liver cancer, hepatocellular carcinoma (HCC), using the orthotopic and subcutaneous tumor model. We found that NK-exo expressed both typical exosomal markers (e.g. CD63, CD81, and Alix) and cytotoxic proteins (e.g. perforin, granzyme B, FasL, and TRAIL). NK-exo were selectively taken up by HCC cells (e.g. Hep3B, HepG2, and Huh 7). Interestingly, Hep3B cells induced the highest cytotoxicity compared with HepG2 and Huh7 cells, and substantially enhanced the apoptosis by NK-exo. Furthermore, we demonstrated that NK-exo inhibited the phosphorylation of serine/threonine protein kinases (e.g. AKT and ERK1/2), and enhanced the activation of specific apoptosis markers (e.g. caspase-3, -7, -8, -9, and PARP) in Hep3B cells. NK-exo also exhibit the active targeting ability and potent therapeutic effects in both orthotopic and subcutaneous HCC mouse models. Overall, these results suggest that NK-exo indicate strong anti-tumor effects in HCC, which are mediated by novel regulatory mechanisms involved in serine/threonine kinase pathway-associated cell proliferation and caspase activation pathway-associated apoptosis. [ABSTRACT FROM AUTHOR]

Oxidized low-density lipoprotein (ox-LDL) stimulation impairs the oxidation-reduction equilibrium in vascular endothelial cells (VECs) and contributes to atherosclerosis (AS). This study probed the mechanisms of extracellular vesicle (EV)-mediated transfer of lncRNA CLDN10 antisense RNA 1 (CLDN10-AS1) in ox-LDL-induced VEC injury. Initially, VEC injury models were established by treating human umbilical vein endothelial cells (HUVECs) with ox-LDL. EVs were isolated from HUVECs (HUVECs-EVs) and identified. CLDN10-AS1, microRNA (miR)-186, and Yin Yang 1 (YY1) expressions in ox-LDL-treated HUVECs and EVs derived from these cells (ox-EVs) were measured. HUVECs were incubated with EVs, after which the cell viability, apoptosis, and concentrations of proinflammatory cytokines and oxidative stress markers were measured. We discovered that CLDN10-AS1 and YY1 were upregulated in ox-LDL-treated HUVECs, whereas miR-186 was downregulated. ox-EVs treatment elevated CLDN10-AS1 expression in HUVECs and ox-EVs overexpressing CLDN10-AS1 promoted VEC injury. Besides, CLDN10-AS1 is competitively bound to miR-186 and promoted YY1 expression. Rescue experiments revealed that miR-186 overexpression or YY1 suppression partially reversed the roles of ox-EVs overexpressing CLDN10-AS1 in ox-LDL-induced VEC injury. Lastly, clinical serum samples were collected for verification. Overall, CLDN10-AS1 carried by HUVECs-EVs into HUVECs competitively bound to miR-186 to elevate YY1 expression, thereby aggravating ox-LDL-induced VEC injury. [ABSTRACT FROM AUTHOR]

T cell‐derived small extracellular vesicles (sEVs) exhibit anti‐cancer effects. However, their anti‐cancer potential should be reinforced to enhance clinical applicability. Herein, we generated interleukin‐2‐tethered sEVs (IL2‐sEVs) from engineered Jurkat T cells expressing IL2 at the plasma membrane via a flexible linker to induce an autocrine effect. IL2‐sEVs increased the anti‐cancer ability of CD8+ T cells without affecting regulatory T (Treg) cells and down‐regulated cellular and exosomal PD‐L1 expression in melanoma cells, causing their increased sensitivity to CD8+ T cell‐mediated cytotoxicity. Its effect on CD8+ T and melanoma cells was mediated by several IL2‐sEV‐resident microRNAs (miRNAs), whose expressions were upregulated by the autocrine effects of IL2. Among the miRNAs, miR‐181a‐3p and miR‐223‐3p notably reduced the PD‐L1 protein levels in melanoma cells. Interestingly, miR‐181a‐3p increased the activity of CD8+ T cells while suppressing Treg cell activity. IL2‐sEVs inhibited tumour progression in melanoma‐bearing immunocompetent mice, but not in immunodeficient mice. The combination of IL2‐sEVs and existing anti‐cancer drugs significantly improved anti‐cancer efficacy by decreasing PD‐L1 expression in vivo. Thus, IL2‐sEVs are potential cancer immunotherapeutic agents that regulate both immune and cancer cells by reprogramming miRNA levels. [ABSTRACT FROM AUTHOR]

Blockade of immune checkpoints as a strategy of cancer cells to overcome the immune response has received ample attention in cancer research recently. In particular, expression of PD‐L1 by various cancer cells has become a paradigm in this respect. Delivery of PD‐L1 to its site of action occurs either by local diffusion, or else by transport via small extracellular vesicles (sEVs, commonly referred to as exosomes). Many steps of sEVs formation, their packaging with PD‐L1 and their release into the extracellular space have been studied in detail. The likely dependence of release on Ca2+‐signaling, however, has received little attention. This is surprising, since the intracellular Ca2+‐concentration is known as a prominent regulator of many secretory processes. Here, we report on the roles of three Ca2+‐dependent proteins in regulating release of PD‐L1‐containing sEVs, as well as on the growth of tumors in mouse models. We show that sEVs release in cancer cell lines is Ca2+‐dependent and the knockdown of the gene coding the Ca2+‐channel protein ORAI1 reduces Ca2+‐signals and release of sEVs. Consequently, the T cell response is reinvigorated and tumor progression in mouse models is retarded. Furthermore, analysis of protein expression patterns in samples from human cancer tissue shows that the ORAI1 gene is significantly upregulated. Such upregulation is identified as an unfavorable prognostic factor for survival of patients with non‐small‐cell lung cancer. We show that reduced Ca2+‐signaling after knockdown of ORAI1 gene also compromises the activity of melanophilin and Synaptotagmin‐like protein 2, two proteins, which are important for correct localization of secretory organelles within cancer cells and their transport to sites of exocytosis. Thus, the Ca2+‐channel ORAI1 and Ca2+‐dependent proteins of the secretion pathway emerge as important targets for understanding and manipulating immune checkpoint blockade by PD‐L1. [ABSTRACT FROM AUTHOR]

Cardiac tissue engineering has emerged as a promising strategy to treat infarcted cardiac tissues by replacing the injured region with an ex vivo fabricated functional cardiac patch. Nevertheless, integration of the transplanted patch with the host tissue is still a burden, limiting its clinical application. Here, a bi-functional, 3D bio-printed cardiac patch (CP) design is proposed, composed of a cell-laden compartment at its core and an extracellular vesicle (EV)-laden compartment at its shell for better integration of the CP with the host tissue. Alginate-based bioink solutions were developed for each compartment and characterized rheologically, examined for printability and their effect on residing cells or EVs. The resulting 3D bio-printed CP was examined for its mechanical stiffness, showing an elastic modulus between 4–5 kPa at day 1 post-printing, suitable for transplantation. Affinity binding of EVs to alginate sulfate (AlgS) was validated, exhibiting dissociation constant values similar to those of EVs with heparin. The incorporation of AlgS-EVs complexes within the shell bioink sustained EV release from the CP, with 88% cumulative release compared with 92% without AlgS by day 4. AlgS also prolonged the release profile by an additional 2 days, lasting 11 days overall. This CP design comprises great potential at promoting more efficient patch assimilation with the host. [ABSTRACT FROM AUTHOR]

Background: Epidemiological studies have identified the role of periodontitis in the pathogenesis of type 2 diabetes, but the underlying mechanism is poorly understood. It is well‐known that small extracellular vesicles are lipid bilayer vesicles derived from cells with a diameter around 30 to 200 nm. The purpose of this study was to investigate whether periodontitis induced or exacerbated insulin resistance via circulating small extracellular vesicles. Methods: Plasma small extracellular vesicles from control and periodontitis rats were intravenously injected into type 2 diabetic rats. Insulin tolerance tests, glucose tolerance tests, and the activation of the insulin signaling pathway were measured to detect the effect of the plasma small extracellular vesicles on insulin sensitivity. In addition, circulating small extracellular vesicles from patients with periodontitis with or without diabetes were isolated and co‐cultured with HepG2 cells. The ability of glucose uptake was assessed using the fluorescence of 2‐NBDG via flow cytometry. The activation of insulin signaling pathway was examined via Western blotting. Real time quantitative polymerase chain reaction (RT‐qPCR) was used to detect the expression of enzyme related to glycolysis and gluconeogenesis. Results: Small extracellular vesicles derived from the plasma of periodontitis rats further impaired glucose tolerance and insulin tolerance in diabetic rats and significantly reduced the activation of the insulin signaling pathway in liver tissues, as evidenced by the decreased levels of p‐AKT and p‐GSK3β and the reduced hepatic glycogen content. For small extracellular vesicles isolated from human plasma, the concentration of small extracellular vesicles in patients with type 2 diabetes combined with periodontitis was higher than that of the healthy control and periodontitis alone. Moreover, circulating small extracellular vesicles from patients with periodontitis significantly inhibited the glucose uptake capacity and inhibited insulin signaling of HepG2 cells. Conclusion: Periodontitis acted as a contributing factor to exacerbate insulin resistance of type 2 diabetic rats. Plasma small extracellular vesicles played a critical role in periodontitis aggravating insulin resistance. [ABSTRACT FROM AUTHOR]

Background: Small extracellular vesicles (sEVs) are emerging natural nanoplatforms in cancer diagnosis and therapy, through the incorporation of signal components or drugs in their structure. However, for their translation into the clinical field, there is still a lack of tools that enable a deeper understanding of their in vivo pharmacokinetics or their interactions with the cells of the tumor microenvironment. In this study, we have designed a dual-sEV probe based on radioactive and fluorescent labeling of goat milk sEVs. Results: The imaging nanoprobe was tested in vitro and in vivo in a model of glioblastoma. In vitro assessment of the uptake of the dual probe in different cell populations (RAW 264.7, U87, and HeLa) by optical and nuclear techniques (gamma counter, confocal imaging, and flow cytometry) revealed the highest uptake in inflammatory cells (RAW 264.7), followed by glioblastoma U87 cells. In vivo evaluation of the pharmacokinetic properties of nanoparticles confirmed a blood circulation time of ~ 8 h and primarily hepatobiliary elimination. The diagnostic capability of the dual nanoprobe was confirmed in vivo in a glioblastoma xenograft model, which showed intense in vivo uptake of the SEV-based probe in tumor tissue. Histological assessment by confocal imaging enabled quantification of tumor populations and confirmed uptake in tumor cells and tumor-associated macrophages, followed by cancer-associated fibroblasts and endothelial cells. Conclusions: We have developed a chemical approach for dual radioactive and fluorescent labeling of sEVs. This methodology enables in vivo and in vitro study of these vesicles after exogenous administration. The dual nanoprobe would be a promising technology for cancer diagnosis and a powerful tool for studying the biological behavior of these nanosystems for use in drug delivery. [ABSTRACT FROM AUTHOR]

The microbiota constitutes an important part of the holobiont in which extracellular vesicles (EVs) are key players in health, especially regarding inter- and intra-kingdom communications. Analysis of EVs from the red blood cell concentrates of healthy donors revealed variable amounts of OmpA and LPS in 12 of the 14 analyzed samples, providing indirect experimental evidence of the presence of microbiota EVs in human circulating blood in the absence of barrier disruption. To investigate the role of these microbiota EVs, we tracked the fusion of fluorescent Escherichia coli EVs with blood mononuclear cells and showed that, in the circulating blood, these EVs interacted almost exclusively with monocytes. This study demonstrates that bacterial EVs constitute critical elements of the host–microbiota cellular communication. The analysis of bacterial EVs should thus be systematically included in any characterization of human EVs. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) in the brain play a role in neuronal homeostasis by removing intracellular material and regulating cell-to-cell communication. Given that sex and aging differentially modulate brain networks, we investigated sex-dependent differences in EV levels and content in the brain during aging. EVs were isolated from the brains of 3, 6, 12, 18, and 24 month-old female and male C57BL/6 J mice, and the levels of different EV species determined. While the number of plasma membrane-derived microvesicles and a subset of late endosomes-derived exosomes increased with age in the brain of female mice, no significant changes were seen in males. Mitochondria-derived mitovesicles in the brain increased during aging in both sexes, a change that may reflect aging-dependent alterations in mitochondrial function. These findings reveal enhanced turnover during aging in female brains, suggesting a mechanism for advantageous successful female brain aging and sex-depending different susceptibility to age-related neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Extracellular vehicles (EVs) are nanoscale lipid bilayer vesicles that carry biologically active biomolecule cargos like proteins, lipids, and nucleic acids (DNA, RNA) outside of the cell. Blood (serum/plasma), urine, and bronchoalveolar lavage fluid are all examples of biofluids from which they may be collected. EVs play a vital role in intracellular communication. The molecular signature of EVs largely depends on the parental cell's status. EVs are classified into two groups, (1) exosomes (originated by endogenous route) and (2) microvesicles (originated from the plasma membrane, also known as ectosomes). The quantity and types of EV cargo vary during normal conditions compared to pathological conditions (chronic inflammatory lung diseases or lung cancer). Consequently, EVs contain novel biomarkers that differ based on the cell type of origin and during lung diseases. Small RNAs (e.g., microRNAs) are transported by EVs, which is one of the most rapidly evolving research areas in the field of EVs biology. EV‐mediated cargos transport small RNAs that can result in reprograming the target/recipient cells. Multiple chronic inflammatory lung illnesses, such as chronic obstructive pulmonary disease, asthma, pulmonary hypertension, pulmonary fibrosis, cystic fibrosis, acute lung injury, and lung cancer, have been demonstrated to be regulated by EV. In this review, we will consolidate the current knowledge and literature on the novel role of EVs and their small RNAs concerning chronic lung diseases (CLDs). Additionally, we will also provide better insight into the clinical and translational impact of mesenchymal stem cells‐derived EVs as novel therapeutic agents in treating CLDs. [ABSTRACT FROM AUTHOR]

To penetrate the zona pellucida before sperm-egg binding, sperm must undergo highly time-controlled capacitation and acrosome reaction in the female reproductive tract. Our previous study demonstrated that miR-21-5p is the most abundant miRNA in boar seminal plasma (SP)-derived extracellular vesicles (EVs) and can target Vinculin (VCL) gene, which may participate in boar sperm capacitation. Thus, this study aims to explore the potential role of miR-21-5p from SP-derived EVs in preventing sperm capacitation and its underlying mechanism. We observed that sperm could incorporate miR-21-5p from SP-derived EVs. The roles of SP-derived EVs miR-21-5p in sperm capacitation were then determined using gain- and loss-of-function analyses. In addition, the expression levels of miR-21-5p, VCL, and VCL protein in liquid-preserved boar sperm following transfection were determined using RT-qPCR and Western blotting. Our results revealed that miR-21-5p overexpression inhibited sperm capacitation and acrosome reaction. Similarly, miR-21-5p expression was significantly lower (P < 0.05) in capacitated sperm than un-capacitated sperm. However, the protein level of VCL was also significantly lower (P < 0.05) in capacitated sperm than un-capacitated sperm. Furthermore, immunofluorescence analysis showed that VCL protein mainly located in sperm head and sperm capacitation was inhibited after treating with VCL protein inhibitor (Chrysin). In conclusion, our study provides reasonable evidence that miR-21-5p expression in SP-derived EVs could prevent sperm capacitation via VCL inhibition. • Sperm could incorporate miR-21-5p from SP-derived EVs. • MiR-21-5p inhibited sperm capacitation and acrosome reaction. • MiR-21-5p was down regulated in boar sperm during sperm capacitation. • VCL protein was required for sperm capacitation. [ABSTRACT FROM AUTHOR]

Extracellular vesicles play a pivotal role in the intercellular communications influencing various physiological and pathological processes. They carry a range of biomolecular cargoes including small non-coding RNAs (ncRNAs) that could serve as potential diagnostic biomarkers and therapeutic targets. In the current study we applied Next Generation Sequencing to investigate small RNA profiles of erythrocytes, platelets, leukocytes, two plasma fractions and blood cell-derived extracellular vesicles (EVs) (CD41+, CD45+, CD235a+, CD146+) obtained using high-sensitivity fluorescence activated vesicle sorting (hs-FAVS) from plasma of healthy donors. We analyzed the proportions of various small ncRNAs across samples and identified sample specific profiles of microRNA (miRNA) and transport RNA-derived fragments (tRFs). It was found that the cumulative small ncRNA profiles of EVs originated from platelets, erythrocytes, and leukocytes, which are considered to be dominant among the vesicles circulating in blood, differ from small ncRNA profiles of plasma fractions, which represent macrovesicles and exosomes in the circulation. The proportion of miRNAs in sorted EVs was significantly lower compared to other samples while the proportion of tRFs was higher. Moreover, all sorted EVs carried mostly cell type non-specific miRNAs. Taken together, the results demonstrate that the combination of hs-FAVS with small-RNA sequencing technique is a powerful approach to analyze small ncRNA profiles in cell specific vesicles. [ABSTRACT FROM AUTHOR]

Objectives: Extracellular vesicles (EVs) are key biological mediators of several physiological functions within the cell microenvironment. Platelets are the most abundant source of EVs in the blood. Similarly, platelet lysate (PL), the best platelet derivative and angiogenic performer for regenerative purposes, is enriched of EVs, but their role is still too poorly discovered to be suitably exploited. Here, we explored the contribution of the EVs in PL, by investigating the angiogenic features extrapolated from that possessed by PL. Methods: We tested angiogenic ability and molecular cargo in 3D bioprinted models and by RNA sequencing analysis of PL‐derived EVs. Results: A subset of small vesicles is highly represented in PL. The EVs do not retain aggregation ability, preserving a low redox state in human umbilical vein endothelial cells (HUVECs) and increasing the angiogenic tubularly‐like structures in 3D endothelial bioprinted constructs. EVs resembled the miRNome profile of PL, mainly enriched with small RNAs and a high amount of miR‐126, the most abundant angiogenic miRNA in platelets. The transfer of miR‐126 by EVs in HUVEC after the in vitro inhibition of the endogenous form, restored angiogenesis, without involving VEGF as a downstream target in this system. Conclusion: PL is a biological source of available EVs with angiogenic effects involving a miRNAs‐based cargo. These properties can be exploited for targeted molecular/biological manipulation of PL, by potentially developing a product exclusively manufactured of EVs. [ABSTRACT FROM AUTHOR]

Doxorubicin (DOXO)-induced cardiomyopathy (DIC) is a lethal complication in cancer patients. Major mechanisms of DIC involve oxidative stress in cardiomyocytes and hyperactivated immune response. Extracellular vesicles (EVs) mediate cell–cell communication during oxidative stress. However, functions of circulating EVs released after chronic DOXO exposure on cardiomyocytes and immune cells are still obscured. Herein, we developed a DIC in vivo model using male Wistar rats injected with 3 mg/kg DOXO for 6 doses within 30 days (18 mg/kg cumulative dose). One month after the last injection, the rats developed cardiotoxicity evidenced by increased BCL2-associated X protein and cleaved caspase-3 in heart tissues, along with N-terminal pro B-type natriuretic peptide in sera. Serum EVs were isolated by size exclusion chromatography. EV functions on H9c2 cardiomyocytes and NR8383 macrophages were evaluated. EVs from DOXO-treated rats (DOXO_EVs) attenuated ROS production via increased glutathione peroxidase-1 and catalase gene expression, and reduced hydrogen peroxide-induced cell death in cardiomyocytes. In contrast, DOXO_EVs induced ROS production, interleukin-6, and tumor necrosis factor-alpha, while suppressing arginase-1 gene expression in macrophages. These results suggested the pleiotropic roles of EVs against DIC, which highlight the potential role of EV-based therapy for DIC with a concern of its adverse effect on immune response. [ABSTRACT FROM AUTHOR]

Embryo–endometrial communication plays a critical role in embryo implantation and the establishment of a successful pregnancy. Successful pregnancy outcomes involve maternal immune modulation during embryo implantation. The endometrium is usually primed and immunomodulated by steroid hormones and embryo signals for subsequent embryo implantation and the maintenance of pregnancy. The roles of extracellular vesicles (EVs) and microRNAs for the embryo–maternal interactions have been elucidated recently. New evidence shows that endometrial EVs and trophectoderm-originated EV cargo, including microRNAs, proteins, and lipids in the physiological microenvironment, regulate maternal immunomodulation for embryo implantation and subsequent pregnancy. On the other hand, trophoblast-derived EVs also control the cross-communication between the trophoblasts and immune cells. The exploration of EV functions and mechanisms in the processes of embryo implantation and pregnancy will shed light on a practical tool for the diagnostic or therapeutic approaches to reproductive medicine and infertility. [ABSTRACT FROM AUTHOR]

Being enucleated, RBCs lack typical transcriptomes, but are known to contain small amounts of diverse long transcripts and microRNAs. However, the exact role and importance of these RNAs are lacking. Shedding of extracellular vesicles (EVs) from the plasma membrane constitutes an integral mechanism of RBC homeostasis, by which RBCs remove unnecessary cytoplasmic content and cell membrane. To study this further, we explored the transcriptomes of RBCs and extracellular vesicles (EVs) of RBCs using next-generation sequencing. Furthermore, we performed single-cell RNA sequencing on RBCs, which revealed that approximately 10% of the cells contained detectable levels of mRNA and cells formed three subpopulations based on their transcriptomes. A decrease in the mRNA quantity was observed across the populations. Qualitative changes included the differences in the globin transcripts and changes in the expression of ribosomal genes. A specific splice form of a long non-coding RNA, Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), was the most enriched marker in one subpopulation of RBCs, co-expressing with ribosomal structural transcripts. MALAT1 expression was confirmed by qPCR in CD71-enriched reticulocytes, which were also characterized with imaging flow cytometry at the single cell level. Analysis of the RBC transcriptome shows enrichment of pathways and functional categories required for the maturation of reticulocytes and erythrocyte functions. The RBC transcriptome was detected in their EVs, making these transcripts available for intercellular communication in blood. [ABSTRACT FROM AUTHOR]

Direct contact of membrane molecules and cytokine interactions orchestrate immune homeostasis. However, overcoming the threshold of distance and velocity barriers, and achieving adhesion mediated immune interaction remain difficult. Here, inspired by the natural chemotaxis of regulatory T cells, multifunctionalized FOXP3 genetic engineered extracellular vesicles, termed Foe‐TEVs, are designed, which display with adhesive molecules, regulatory cytokines, and coinhibitory contact molecules involving CTLA‐4 and PD‐1, by limited exogenous gene transduction. Foe‐TEVs effectively adhere to the tubular, endothelial, and glomerular regions of allogeneic injury in the renal allograft, mitigating cell death in situ and chronic fibrosis transition. Remarkably, transcript engineering reverses the tracking velocity of vesicles to a retained phenotype and enhanced arrest coefficient by a factor of 2.16, directly interacting and attenuating excessive allosensitization kinetics in adaptive lymphoid organs. In murine allogeneic transplantation, immune adhesive Foe‐TEVs alleviate pathological responses, restore renal function with well ordered ultrastructure and improved glomerular filtration rate, and prolong the survival period of the recipient from 30.16 to 92.81 days, demonstrating that the delivery of extracellular vesicles, genetically engineered for immune adhesive, is a promising strategy for the treatment of graft rejection. [ABSTRACT FROM AUTHOR]

Objectives: To develop a modified Vesical Imaging Reporting and Data System (VI-RADS) without dynamic contrast-enhanced imaging (DCEI), termed "non-contrast-enhanced VI-RADS (NCE-VI-RADS)", and to assess the additive impact of denoising deep learning reconstruction (dDLR) on NCE-VI-RADS. Methods: From January 2019 through December 2020, 163 participants who underwent high-gradient 3-T MRI of the bladder were prospectively enrolled. In total, 108 participants with pathologically confirmed bladder cancer by transurethral resection were analyzed. Tumors were evaluated based on VI-RADS (scores 1–5) by two readers independently: an experienced radiologist (reader 1) and a senior radiology resident (reader 2). Conventional VI-RADS assessment included all three imaging types (T2-weighted imaging [T2WI], diffusion-weighted imaging [DWI], and dynamic contrast-enhanced imaging [DCEI]). Also evaluated were NCE-VI-RADS comprising only non-contrast-enhanced imaging types (T2WI and DWI), and "NCE-VI-RADS with dDLR" comprising T2WI processed with dDLR and DWI. All systems were assessed using receiver-operating characteristic curve analysis and simple and/or weighted κ statistics. Results: Muscle invasion was identified in 23/108 participants (21%). Area under the curve (AUC) values for diagnosing muscle invasion were as follows: conventional VI-RADS, 0.94 and 0.91; NCE-VI-RADS, 0.93 and 0.91; and "NCE-VI-RADS with dDLR", 0.96 and 0.93, for readers 1 and 2, respectively. Simple κ statistics indicated substantial agreement for NCE-VI-RADS and almost perfect agreement for conventional VI-RADS and "NCE-VI-RADS with dDLR" between the two readers. Conclusion: NCE-VI-RADS achieved predictive accuracy for muscle invasion comparable to that of conventional VI-RADS. Additional use of dDLR improved the diagnostic accuracy of NCE-VI-RADS. Key Points: • Non-contrast-enhanced Vesical Imaging Reporting and Data System (NCE-VI-RADS) was developed to avoid risk related to gadolinium-based contrast agent administration. • NCE-VI-RADS had predictive accuracy for muscle invasion comparable to that of conventional VI-RADS. • The additional use of denoising deep learning reconstruction (dDLR) might further improve the diagnostic accuracy of NCE-VI-RADS. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs), including exosomes and microvesicles, are released by numerous cell types. EVs are now acknowledged as playing a critical role in cell–cell communication in healthy aging as well as in age‐related diseases. Recently it was shown that senescence, a key hallmark of aging, increases the secretion of EVs. Moreover, EVs can transport proteins and microRNAs (miRNAs) that are key components of the senescence‐associated secretory phenotype (SASP). Here we review evidence that SASP‐related miRNAs are involved in musculoskeletal degeneration with aging. Specifically, senescence‐related miRNAs are elevated in EVs released by skeletal muscle myocytes and fibro‐adipogenic progenitor cells with aging and disuse atrophy, respectively. Many of these same senescence‐related miRNAs are detected in EVs from the synovial fluid of patients with osteoarthritis, and these miRNAs can contribute to cartilage degeneration. Finally, senescence‐associated miRNAs are secreted from bone marrow–derived stem (stromal) cells impacting neighboring hematopoietic stem cells and circulating in the blood. The senescence‐associated miRNA mir‐34a, which is known to target Wnt and Notch pathways as well as the cell survival factors Sirt1 and Bcl2, is detected in EVs from human and animal subjects with muscle atrophy, bone loss, and osteoarthritis. These findings suggest that suppressing the secretion of EV‐derived, senescence‐related miRNAs, such as miR‐34a, or increasing levels of competing endogenous long noncoding RNAs, such as MALAT1 that inhibit miR‐34a, may help to improve musculoskeletal function with aging. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a degenerative joint disease with inadequately understood pathogenesis leading to pain and functional limitations. Extracellular vesicles (EVs) released by synovial joint cells can induce both pro- and anti-OA effects. Hyaluronic acid (HA) lubricates the surfaces of articular cartilage and is one of the bioactive molecules transported by EVs. In humans, altered EV counts and composition can be observed in OA synovial fluid (SF), while EV research is in early stages in the horse—a well-recognized OA model. The aim was to characterize SF EVs and their HA cargo in 19 horses. SF was collected after euthanasia from control, OA, and contralateral metacarpophalangeal joints. The SF HA concentrations and size distribution were determined with a sandwich-type enzyme-linked sorbent assay and size-exclusion chromatography. Ultracentrifugation followed by nanoparticle tracking analysis (NTA) were utilized to quantify small EVs, while confocal laser scanning microscopy (CLSM) and image analysis characterized larger EVs. The number and size distribution of small EVs measured by NTA were unaffected by OA, but these results may be limited by the lack of hyaluronidase pre-treatment of the samples. When visualized by CLSM, the number and proportion of larger HA-containing EVs (HA–EVs) decreased in OA SF (generalized linear model, count: p = 0.024, %: p = 0.028). There was an inverse association between the OA grade and total EV count, HA–EV count, and HA–EV % (rs = – 0.264 to – 0.327, p = 0.012–0.045). The total HA concentrations were also lower in OA (generalized linear model, p = 0.002). To conclude, the present study discovered a potential SF biomarker (HA–EVs) for naturally occurring equine OA. The roles of HA–EVs in the pathogenesis of OA and their potential as a joint disease biomarker and therapeutic target warrant future studies. [ABSTRACT FROM AUTHOR]

The pathogenesis of Alzheimer's disease (AD) remains unknown till today, hindering the research and development of AD therapeutics and diagnostics. Circulating extracellular vesicles (EVs) can be utilized as a new window to spy upon AD pathogenesis. Altered microRNA profiles were noted in both the cerebrospinal fluid (CSF)- and blood-isolated EVs of AD patients, implying the outstanding potential of circulating EV-containing miRNAs (CEmiRs) to serve as important regulators in AD pathogenesis. Although several CEmiRs were found to play a part in AD, the association of globally altered miRNA profiles in patients' serum-derived EVs with AD pathogenesis remains unclear. In this study, we first investigated the miRNA profile in serum-derived EVs from AD, mild cognitive impairment (MCI) patients, and healthy individuals. We observed differential expression patterns of CEmiRs and classified them into 10 clusters. We identified the predicted targets of these differentially expressed CEmiRs (DECEmiRs) and analyzed their biological functions and interactions. Our study revealed the temporal regulation of complex and precise signaling networks on AD pathogenesis, shedding light on the development of novel therapeutic strategies, including multi-target drug combination for AD treatment. [ABSTRACT FROM AUTHOR]

Minimal hepatic encephalopathy (MHE) is associated with changes in the immune system including an increased pro-inflammatory environment and altered differentiation of CD4+ T lymphocytes. The mechanisms remain unknown. Changes in extracellular vesicle (EV) cargo including proteins and miRNAs could play a main role as mediators of immune system changes associated with MHE. The aim was to assess whether plasma EVs from MHE patients played a role in inducing the pro-inflammatory environment and altered differentiation of CD4+ T lymphocyte subtypes in MHE patients. We characterized the miRNA and protein cargo of plasma EVs from 50 cirrhotic patients (27 without and 23 with MHE) and 24 controls. CD4+ T cells from the controls were cultured with plasma EVs from the three groups of study, and the cytokine release and differentiation to CD4+ T-cell subtypes were assessed. Plasma EVs from MHE patients had altered miRNA and protein contents, and were enriched in inflammatory factors compared to the controls and patients without MHE. EVs from MHE patients modulated the expression of pro-inflammatory IL-17, IL-21, and TNF-α and anti-inflammatory TGF-β in cultured CD4+ T lymphocytes, and increased the proportion of Th follicular and Treg cells and the activation of Th17 cells. In conclusion, plasma EVs could play an important role in the induction of immune changes observed in MHE. [ABSTRACT FROM AUTHOR]

Goldfish are one of the most popular models for studying the genetic diversity of skin color. Transcriptome sequencing (RNA-seq) and whole genome bisulfate sequencing (WGBS) of skin tissues from the third filial (F3) cyan (CN), black (BK), and white (WH) goldfish were conducted to analyze the molecular mechanism of color transformation in fish. The RNA-seq yielded 56 Gb of clean data and 56,627 transcripts from nine skin samples. The DEGs (differentially expressed genes) were enriched in cell junction cellular components and the tight junction pathway. Ninety-five homologs of the claudin family were predicted and 16 claudins were identified in correlation with skin color transformation. WGBS yielded 1079 Gb of clean data from 15 samples. Both the DEGs and the DMRs (differentially methylated regions) in the BK_CN group were found to be enriched in cytoskeleton reorganization and vesicle trafficking. Masson staining and TEM (transmission electron microscopy) confirmed the varied distribution and processes of melanosome/melanin in skin tissues. Our results suggested that cytoskeleton reorganization, cell junction, and the vesicle trafficking system played key roles in the transfer of the melanosome/melanin, and it was the extracellular translocation rather than the biosynthesis or metabolism of the melanin process that resulted in the color transformation of cyan goldfish. The data will facilitate the understanding of the molecular mechanisms underlying dynamic skin color transformation in goldfish. [ABSTRACT FROM AUTHOR]