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In recent years, extracellular vesicles (EVs) have become a subject of intense study. These membrane-enclosed spherical structures are secreted by almost every cell type and are engaged in the transport of cellular content (cargo) from parental to target cells. The impact of EVs transfer has been observed in many vital cellular processes including cell-to-cell communication and immune response modulation; thus, a fast and precise characterization of EVs may be relevant for both scientific and diagnostic purposes. In this review, the most popular analytical techniques used in EVs studies are presented with the emphasis on exosomes and microvesicles characterization.

Background Human adipose stem cells (ASCs) have emerged as a promising treatment paradigm for skin wounds. Recent works demonstrate that the therapeutic effect of stem cells is partially mediated by extracellular vesicles, which comprise exosomes and microvesicles. In this study, we investigate the regenerative effects of isolated microvesicles from ASCs and evaluate the mechanisms how ASC microvesicles promote wound healing. Methods Adipose stem cell-derived microvesicles (ASC-MVs) were isolated by differential ultracentrifugation, stained by PKH26, and characterized by electron microscopy and dynamic light scattering (DLS). We examined ASC-MV effects on proliferation, migration, and angiogenesis of keratinocytes, fibroblasts, and endothelial cells both in vitro and in vivo. Next, we explored the underlying mechanisms by gene expression analysis and the activation levels of AKT and ERK signaling pathways in all three kinds of cells after ASC-MV stimulation. We then assessed the effect of ASC-MVs on collagen deposition, neovascularization, and re-epithelialization in an in vivo skin injury model. Results ASC-MVs could be readily internalized by human umbilical vein endothelial cells (HUVECs), HaCAT, and fibroblasts and significantly promoted the proliferation, migration, and angiogenesis of these cells both in vitro and in vivo. The gene expression of proliferative markers (cyclin D1, cyclin D2, cyclin A1, cyclin A2) and growth factors (VEGFA, PDGFA, EGF, FGF2) was significantly upregulated after ASC-MV treatment. Importantly, ASC-MVs stimulated the activation of AKT and ERK signaling pathways in those cells. The local injection of ASC-MVs at wound sites significantly increased the re-epithelialization, collagen deposition, and neovascularization and led to accelerated wound closure. Conclusions Our data suggest that ASC-MVs can stimulate HUVEC, HaCAT, and fibroblast functions. ASC-MV therapy significantly accelerates wound healing, and the benefits of ASC-MVs may due to the involvement of AKT and ERK signaling pathways. This illustrates the therapeutic potential of ASC-MVs which may become a novel treatment paradigm for cutaneous wound healing.

Exosomes and microvesicles (EMV) are lipid bilayer-enclosed structures, released by cells and involved in intercellular communication through transfer of proteins and genetic material. EMV release is also associated with various pathologies, including cancer, where increased EMV release is amongst other associated with chemo-resistance and active transfer of pro-oncogenic factors. Recent studies show that EMV-inhibiting agents can sensitize cancer cells to chemotherapeutic agents and reduce cancer growth . Cannabidiol (CBD), a phytocannabinoid derived from , has anti-inflammatory and anti-oxidant properties, and displays anti-proliferative activity. Here we report a novel role for CBD as a potent inhibitor of EMV release from three cancer cell lines: prostate cancer (PC3), hepatocellular carcinoma (HEPG2) and breast adenocarcinoma (MDA-MB-231). CBD significantly reduced exosome release in all three cancer cell lines, and also significantly, albeit more variably, inhibited microvesicle release. The EMV modulating effects of CBD were found to be dose dependent (1 and 5 μM) and cancer cell type specific. Moreover, we provide evidence that this may be associated with changes in mitochondrial function, including modulation of STAT3 and prohibitin expression, and that CBD can be used to sensitize cancer cells to chemotherapy. We suggest that the known anti-cancer effects of CBD may partly be due to the regulatory effects on EMV biogenesis, and thus CBD poses as a novel and safe modulator of EMV-mediated pathological events.

Background Premature ovarian insufficiency (POI) is one of the leading causes of female infertility, which is caused by an abnormal ovarian reserve. Currently, there is no effective treatment to restore the fertility of POI patients. Recent studies suggested that microvesicles (MVs) released from mesenchymal stem cells (MSCs) exert therapeutic effects in various degenerative diseases. In this study, the effect of human umbilical cord MSC-derived MVs (HUCMSC-MVs) on the restoration of ovarian function in a chemotherapy-induced POI mouse model is investigated. Methods MVs were obtained from the supernatant of cultured HUCMSCs. The localization of PKH26-labeled HUCMSC-MVs in ovarian tissues was observed by confocal laser scanning microscopy. Histomorphometric analysis was performed to count the number of ovarian follicles and vessels. The ovarian sections were stained with anti-CD34 to evaluate angiogenesis. The levels of estradiol (E2) and follicle-stimulating hormone (FSH) were measured by enzyme-linked immunosorbent serologic assay. The mRNA expression of angiogenesis-related cytokines and the protein expression of AKT in mouse ovaries were measured by quantitative RT-PCR and western blot analysis. The parametric variables were compared by Student’s t test and analysis of variance. The non-parametric variables were compared by the Mann-Whitney U test. Categorical variables were compared by χ 2 test. P  < 0.05 was considered statistically significant. Results PKH26-labeled HUCMSC-MVs were detectable within the ovaries and migrated to the ovarian follicles 24 h after transplantation. The transplantation of HUCMSC-MVs could increase the body weight and number of ovarian follicles (primordial, developing, and preovulatory follicles), induce ovarian angiogenesis, and recover the disturbed estrous cycle of POI mice. The expression levels of total AKT, p-AKT, and angiogenic cytokines (including VEGF, IGF, and angiogenin) in the ovaries of POI mice were markedly upregulated after HUCMSC-MVs transplantation, suggesting that HUCMSC-MVs transplantation might recover ovarian function by inducing angiogenesis via the PI3K/AKT signaling pathway. Conclusions This study provides valuable insight into the effects of HUCMSC-MVs on ovarian tissue angiogenesis and on the restoration of ovarian function in POI mice, which may be helpful to develop a treatment strategy for POI patients.

Diabetic nephropathy (DN) is a significant complication of diabetes and is characterized by progressive kidney damage and dysfunction. Several studies have highlighted the role of a subset of large-sized extracellular vesicles (EVs), commonly known as microvesicles (MVs), as crucial mediators of DN pathophysiology. This systematic review critically evaluates the methodological approaches used to study MVs in experimental models of DN, while also synthesizing the experimental endpoints investigated, to identify consistencies, gaps, and opportunities for standardization. A systematic literature search across PubMed, Embase, and Scopus identified preclinical studies investigating the impact of MVs on renal injury, inflammation, and fibrosis in diabetes. Seven preclinical studies published between 2014 and 2022 met the inclusion criteria. Data extracted: MV origin, isolation/characterization/quantification, models/conditions, dosing/exposure, and endpoints. Seven studies (2014–2022) met criteria. Differential centrifugation predominated for isolation; flow cytometry (FCM) (often Annexin V ± lineage markers), nanoparticle tracking analysis (NTA), and electron microscopy (EM) variably supported identity/size; FCM and NTA were commonly used for enumeration along with protein assays. MV sources included platelets, podocytes, urinary fractions, and MSC-derived vesicles. Across studies, MVs modulated oxidative stress (NOX4/ROS), inflammation (e.g., TNF-α, CXCL7), fibrotic signaling (p38 MAPK/CD36), and cell injury; cargo (e.g., miR-451a) linked to cell-cycle regulators (p15/p19) in early DN. Notable heterogeneity in media depletion, dose reporting, and detection thresholds limited cross-study comparison. We conclude that preclinical evidence supports MVs as early biomarkers and mechanistic drivers in DN, but standardization in isolation, characterization, dosing, and endpoint panels—aligned with MISEV 2023—is needed to enable comparability and translation.

Atherosclerosis and cardiovascular disease development is the outcome of intermediate processes where endothelial dysfunction and vascular inflammation are main protagonists. Cell-derived microvesicles (MVs), endothelial progenitor cells (EPCs), and circulating microRNAs (miRNAs) are known as biomarkers and potential regulators for atherosclerotic vascular disease, but their role in the complexity of the inflammatory process and in the mechanism of vascular restoration is far from clear. We aimed to evaluate the biological activity and functional role of MVs, in particular of the EPCs-derived MVs (MVEs), of healthy origins in reducing atherosclerotic vascular disease development. The experiments were performed on hamsters divided into the following groups: simultaneously hypertensive–hyperlipidemic (HH group) by combining two feeding conditions for 4 months; HH with retro-orbital sinus injection containing 1 × 105 MVs or MVEs from control hamsters, one dose per month for 4 months of HH diet, to prevent atherosclerosis (HH-MVs or HH-MVEs group); and controls (C group), age-matched normal healthy animals. We found that circulating MV and MVE transplantation of healthy origins significantly reduces atherosclerosis development via (1) the mitigation of dyslipidemia, hypertension, and circulating EPC/cytokine/chemokine levels and (2) the structural and functional remodeling of arterial and left ventricular walls. We also demonstrated that (1) circulating MVs contain miRNAs; this was demonstrated by validating MVs and MVEs as transporters of Ago2-miRNA, Stau1-miRNA, and Stau2-miRNA complexes and (2) MV and MVE administration significantly protect against atherosclerotic cardiovascular disease via transfer of miR-223, miR-21, miR-126, and miR-146a to circulating late EPCs. It should be mentioned that the favorable effects of MVEs were greater than those of MVs. Our findings suggest that allogenic MV and MVE administration of healthy origins could counteract HH diet-induced detrimental effects by biologically active miR-10a, miR-21, miR-126, and miR-146a transfer to circulating EPCs, mediating their vascular repair function in atherosclerosis processes.

Background Our previous study found that bone marrow-derived mesenchymal stem cells (BMSCs) promote Helicobacter pylori (H pylori) -associated gastric cancer (GC) progression by secreting thrombospondin-2 (THBS2). Extracellular vesicles (EVs) are important carriers for intercellular communication, and EVs secreted by BMSCs have been shown to be closely related to tumor development. The aim of this study was to investigate whether BMSC-derived microvesicles (MVs, a main type of EV) play a role in H. pylori -associated GC by transferring THBS2. Methods BMSCs and THBS2-deficient BMSCs were treated with or without the supernatant of H. pylori for 12 h at a multiplicity of infection of 50, and their EVs were collected. Then, the effects of BMSC-derived MVs and THBS2-deficient BMSC-derived MVs on the GC cell line MGC-803 were assessed by in vitro proliferation, migration, and invasion assays. In addition, a subcutaneous xenograft tumor model, a nude mouse intraperitoneal metastasis model, and a tail vein injection metastasis model were constructed to evaluate the effects of BMSC-derived MVs and THBS2-deficient BMSC-derived MVs on GC development and metastasis in vivo. Results BMSC-derived MVs could be readily internalized by MGC-803 cells. BMSC-derived MVs after H. pylori treatment significantly promoted their proliferation, migration and invasion in vitro (all P  < 0.05) and promoted tumor development and metastasis in a subcutaneous xenograft tumor model, a nude mouse intraperitoneal metastasis model, and a tail vein injection metastasis model in vivo (all P  < 0.05). The protein expression of THBS2 was significantly upregulated after H. pylori treatment in BMSC-derived MVs ( P  < 0.05). Depletion of the THBS2 gene reduces the tumor-promoting ability of BMSC-MVs in an H. pylori infection microenvironment both in vitro and in vivo. Conclusion Overall, these findings indicate that MVs derived from BMSCs can promote H. pylori -associated GC development and metastasis by delivering the THBS2 protein. 16ZaMehzoqZcatB8td6PJe Video Abstract

Exosomes and microvesicles (EMVs) are lipid bilayer-enclosed structures released from cells and participate in cell-to-cell communication via transport of biological molecules. EMVs play important roles in various pathologies, including cancer and neurodegeneration. The regulation of EMV biogenesis is thus of great importance and novel ways for manipulating their release from cells have recently been highlighted. One of the pathways involved in EMV shedding is driven by peptidylarginine deiminase (PAD) mediated post-translational protein deimination, which is calcium-dependent and affects cytoskeletal rearrangement amongst other things. Increased PAD expression is observed in various cancers and neurodegeneration and may contribute to increased EMV shedding and disease progression. Here, we review the roles of PADs and EMVs in cancer and neurodegeneration.

Platelets and their extracellular vesicles (EVs) have emerged as promising liquid biopsy biosources for cancer detection and monitoring. The megakaryoblastic MEG-01 cell line offers a controlled system for generating platelet-like particles (PLPs) and EVs through valproic-acid-induced differentiation. Here, we performed comprehensive characterization and proteomic validation of MEG-01-derived populations, native human platelets, and their EVs using nanoparticle tracking analysis, transmission electron microscopy, imaging flow cytometry and quantitative proteomics. MEG-01 megakaryocytic differentiation is characterized by polylobulated nuclei, proplatelet formation, and elevated CD41/CD42a expression. PLPs predominantly exhibit an activated-like phenotype (CD62P+, degranulated morphology), while microvesicles (100-500 nm) and exosomes (50-250 nm) displayed size distributions and phenotypic markers consistent with native platelet-derived EVs. Proteomics identified substantial core proteomes shared across fractions and fraction-specific patterns consistent with selective cargo partitioning during EV biogenesis. Functional enrichment indicated that MEG-01-derived vesicles preserve key hemostatic, cytoskeletal, and immune pathways commonly associated with platelet EV biology. Ingenuity Pathway Analysis showed that PLPs exhibit proliferative transcriptional programs (elevated MYC/RB1/TEAD1, reduced GATA1), while plasma exosomes display minimal differential pathway activation compared to MEG-01 exosomes. Overall, these findings suggest that MEG-01-derived EVs approximate certain aspects of megakaryocyte-lineage exosomes and activated platelet-like states, although they do not fully replicate native platelet biology. Notably, plasma exosomes show strong proteomic convergence with MEG-01 exosomes, whereas platelet exosomes retain distinct activation-related features.