Small extracellular vesicles (sEV) are endogenous lipid-bound membrane vesicles secreted by both prokaryotic and eukaryotic cells into the extracellular environment, performs several biological functions such as cell-cell communication, transfer of proteins, mRNA, and ncRNA to target cells in distant sites. Due to their role in molecular pathogenesis and its potential to deliver biological cargo to target cells, it has become a prominent area of interest in recent research in the field of Neuroscience. However, their role in neurological disorders, like neurodegenerative diseases is more complex and still unaddressed. Thus, this review focuses on the role of sEV in neurodegenerative and neurodevelopmental diseases, including their biogenesis, classification, and pathogenesis, with translational advantages and limitations in the area of neurobiology.

Modulating the inflammatory microenvironment to reconstruct the fibrocartilaginous layer while promoting tendon repair is crucial for enhancing tendon-to-bone healing in rotator cuff repair (RCR), a persistent challenge in orthopedics. Small extracellular vesicles (sEVs) hold significant potential to modulate inflammation, yet the efficient production of highly bioactive sEVs remains a substantial barrier to their clinical application. Moreover, achieving minimally invasive local delivery of sEVs to the tendon-to-bone interface presents significant technical difficulties. Herein, the circadian rhythm of adipose-derived stem cells is modulated to increase the yield and enhance the inflammatory regulatory capacity of sEVs. Circadian rhythm-regulated sEVs (CR-sEVs) enhance the cyclic adenosine monophosphate signaling pathway in macrophage (Mφ) via platelet factor 4 delivery, thereby inhibiting Mφ M1 polarization. Subsequently, a triphasic microneedle (MN) scaffold with a tip, stem, and base is designed for the local delivery of CR-sEVs (CR-sEVs/MN) at the tendon-to-bone junction, incorporating tendon-derived decellularized extracellular matrix in the base to facilitate tendon repair. CR-sEVs/MN mitigates inflammation, promotes fibrocartilage regeneration, and enhances tendon healing, thereby improving biomechanical strength and shoulder joint function in a rat RCR model. Combining CR-sEVs with this triphasic microneedle delivery system presents a promising strategy for enhancing tendon-to-bone healing in clinical settings.

It is known that small extracellular vesicles (sEVs) are released from cancer cells and contribute to cancer progression via crosstalk with recipient cells. We have previously reported that sEVs expressing the αVβ3 integrin, a protein upregulated in aggressive neuroendocrine prostate cancer (NEPrCa), contribute to neuroendocrine differentiation (NED) in recipient cells. Here, we examine the impact of αVβ3 expression on sEV protein content, density and function. sEVs used in this study were isolated by iodixanol density gradients and characterized by nanoparticle tracking analysis, immunoblotting and single vesicle analysis. Our proteomic profile of sEVs containing αVβ3 shows downregulation of typical effectors involved in apoptosis and necrosis and an upregulation of tumour cell survival factors compared to control sEVs. We also show that the expression of αVβ3 in sEVs causes a distinct reposition of EV markers (Alix, CD81, CD9) to a low-density sEV subpopulation. This low-density reposition is independent of extracellular matrix (ECM) protein interactions with sEVs. This sEV subset contains αVβ3 and an αVβ3 downstream effector, NgR2, a novel marker for NEPrCa. We show that sEVs containing αVβ3 are loaded with higher amounts of NgR2 as compared to sEVs that do not express αVβ3. Mechanistically, we demonstrate that sEVs containing NgR2 do not affect the sEV marker profile, but when injected in vivo intratumorally, they promote tumour growth and induce NED. We show that sEVs expressing NgR2 increase the activation of focal adhesion kinase (FAK), a known promoter of cancer cell proliferation, in recipient cells. We also show that NgR2 mimics the effect of sEVs containing αVβ3 since it displays increased growth of NgR2 transfectants in vivo, as compared to control cells. Overall, our results describe the changes that occur in cargo, density and functions of cancer cell-derived sEVs containing the αVβ3 integrin and its effector, NgR2, without affecting the sEV tetraspanin profiles.

Engineering of extracellular vesicles (EVs) towards more efficient targeting and uptake to specific cells has large potentials for their application as therapeutics. Carbohydrates play key roles in various biological interactions and are essential for EV biology. The extent to which glycan modification of EVs can be achieved through genetic glycoengineering of their parental cells has not been explored yet. Here we introduce targeted glycan modification of EVs through cell-based glycoengineering via modification of various enzymes in the glycosylation machinery. In a \"simple cell\" strategy, we modified major glycosylation pathways by knocking-out (KO) essential genes for N-glycosylation (MGAT1), O-GalNAc glycosylation (C1GALT1C1), glycosphingolipids (B4GALT5/6), glycosaminoglycans (B4GALT7) and sialylation (GNE) involved in the elongation or biosynthesis of the glycans in HEK293F cells. The gene editing led to corresponding glycan changes on the cells as demonstrated by differential lectin staining. Small EVs (sEVs) isolated from the cells showed overall corresponding glycan changes, but also some unexpected differences to their parental cell including enrichment preference for certain glycan structures and absence of other glycan types. The genetic glycoengineering did not significantly impact sEVs production, size distribution, or syntenin-1 biomarker expression, while a clonal influence on sEVs production yields was observed. Our findings demonstrate the successful implementation of sEVs glycoengineering via genetic modification of the parental cell and a stable source for generation of glycoengineered sEVs. The utilization of glycoengineered sEVs offers a promising opportunity to study the role of glycosylation in EV biology, as well as to facilitate the optimization of sEVs for therapeutic purposes.

Evidence suggests that immune system dysfunction and macrophages are involved in the disease establishment and progression of endometriosis. Among the factors involved in this alteration in macrophage activity, Small Extracellular Vesicles (sEVs) have been described to play a role favoring the switch to a specific phenotype with controversial results. This study aims to investigate the potential effect of circulating sEVs in the plasma of well-characterized patients with endometriosis on the polarization of macrophages. sEVs were isolated from the plasma of patients diagnosed with endometriosis confirmed by histopathological analysis. Two groups of patients were recruited: the endometriosis group consisted of patients diagnosed with endometriosis by imaging testing (gynecological ultrasonography and/or magnetic resonance imaging), confirmed by histopathologic study (n = 12), and the control group included patients who underwent laparoscopy for tubal sterilization without presurgical suspicion of endometriosis and without endometriosis or signs of any inflammatory pelvic condition during surgery (n = 12). Human THP1 monocytic cells were differentiated into macrophages, and the effect of sEVs on cell uptake and macrophage polarization was evaluated by fluorescent labeling and measurement of the IL1B, TNF, ARG1, and MRC1 expression, respectively. Although no changes in cell uptake were detected, sEVs from endometriosis induced a polarization of macrophages toward an M2 phenotype, characterized by lower IL1B and TNF expression and a tendency to increase MRC1 and ARG1 levels. When macrophages were stimulated with lipopolysaccharides, less activation was also detected after treatment with endometriosis sEVs. Finally, endometriosis sEVs also induced the expression of the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARG); however, treatment with rosiglitazone, a PPARG agonist, had no effect on the change in macrophage phenotype. We conclude that circulating sEVs in women with endometriosis have a certain capacity to shift the activation state of macrophages toward an M2 phenotype, but this does not modify the uptake level or the response to PPARG ligands.

(Background). Canine mammary tumors (CMTs) have emerged as an important model for understanding pathophysiological aspects of human disease. Liquid biopsy (LB), which relies on blood-borne biomarkers and offers minimal invasiveness, holds promise for reflecting the disease status of patients. Small extracellular vesicles (SEVs) and their protein cargo have recently gained attention as potential tools for disease screening and monitoring. (Objectives). This study aimed to isolate SEVs from canine patients and analyze their proteomic profile to assess their diagnostic and prognostic potential. (Methods). Plasma samples were collected from female dogs grouped into CMT (malignant and benign), healthy controls, relapse, and remission groups. SEVs were isolated and characterized using ultracentrifugation (UC), nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Proteomic analysis of circulating SEVs was conducted using liquid chromatography-mass spectrometry (LC-MS). (Results). While no significant differences were observed in the concentration and size of exosomes among the studied groups, proteomic profiling revealed important variations. Mass spectrometry identified exclusive proteins that could serve as potential biomarkers for mammary cancer. These included Inter-alpha-trypsin inhibitor heavy chain (ITIH2 and ITI4), phosphopyruvate hydratase or alpha enolase (ENO1), eukaryotic translation elongation factor 2 (eEF2), actin (ACTB), transthyretin (TTR), beta-2-glycoprotein 1 (APOH) and gelsolin (GSN) found in female dogs with malignant tumors. Additionally, vitamin D-binding protein (VDBP), also known as group-specific component (GC), was identified as a protein present during remission. (Conclusions). The results underscore the potential of proteins found in SEVs as valuable biomarkers in CMTs. Despite the lack of differences in vesicle concentration and size between the groups, the analysis of protein content revealed promising markers with potential applications in CMT diagnosis and monitoring. These findings suggest a novel approach in the development of more precise and effective diagnostic tools for this challenging clinical condition.

Prostate cancer is a significant health problem in the United States. It is remarkably heterogenous, ranging from slow growing disease amenable to active surveillance to highly aggressive forms requiring active treatments. Therefore, being able to precisely determine the nature of disease and appropriately match patients to available and/or novel therapeutics is crucial to improve patients\' overall outcome and quality of life. Recently small extracellular vesicles (sEVs), a subset of nanoscale membranous vesicles secreted by various cells, have emerged as important analytes for liquid biopsy and promising vehicles for drug delivery. sEVs contain various biomolecules such as genetic material, proteins, and lipids that recapitulate the characteristics and state of their donor cells. The application of existing and newly developed technologies has resulted in an increased depth of knowledge about biophysical structures, biogenesis, and functions of sEVs. In prostate cancer patients, tumor-derived sEVs can be isolated from biofluids, commonly urine and blood. They mediate intercellular signaling within the tumor microenvironment and distal organ-specific sites, supporting cancer initiation, progression, and metastasis. A mounting body of evidence suggests that sEV components can be potent biomarkers for prostate cancer diagnosis, prognosis, and prediction of disease progression and treatment response. Due to enhanced circulation stability and bio-barrier permeability, sEVs can be also used as effective drug delivery carriers to improve the efficacy and specificity of anti-tumor therapies. This review discusses recent studies on sEVs in prostate cancer and is focused on their role as biomarkers and drug delivery vehicles in the clinical management of prostate cancer.

Human umbilical cord mesenchymal stem cells-derived small extracellular vesicles (MSC-sEV) provide a pragmatic solution as a cell-free therapy for patients with diabetic kidney disease (DKD). However, the underlying protective mechanisms of MSC-sEV remain largely unknown in DKD. Invivo and in vitro analyses demonstrated that MSC-sEV attenuated renal fibrosis and inflammation of DKD. The underlying mechanism of the MSC-sEV-induced therapeutic effect was explored by high-throughput sequencing, which identified the unique enrichment of a set of miRNAs in MSC-sEV compared with human skin fibroblasts-sEV (HSF-sEV). Vitro experiments demonstrated that the protective potential was primarily attributed to miR-23a-3p, one of the most abundant miRNAs in MSC-sEV. Further, overexpression or knockdown analyses revealed that miR-23a-3p, and its target Krüppel-like factor 3 (KLF3) suppressed the STAT3 signaling pathway in high glucose (HG) induced HK-2 cells were essential for the renal-protective property of MSC-sEV. Moreover, we found that miR-23a-3p was packaged into MSC-sEV by RNA Binding Motif Protein X-Linked (RBMX) and transmitted to HG-induced HK-2 cells. Finally, inhibiting miR-23a-3p could mitigate the protective effects of MSC-sEV in db/db mice. These findings suggest that a systemic administration of sEV derived from MSC, have the capacity to incorporate into kidney where they can exert renal-protective potential against HG-induced injury through delivery of miR-23a-3p.

Tissues release microRNAs (miRNAs) in small extracellular vesicles (sEVs) including exosomes, which can regulate gene expression in distal cells, thus acting as modulators of local and systemic metabolism. Here, we show that insulin regulates miRNA secretion into sEVs from 3T3-L1 adipocytes and that this process is differentially regulated from cellular expression. Thus, of the 53 miRNAs upregulated and 66 miRNAs downregulated by insulin in 3T3-L1 sEVs, only 12 were regulated in parallel in cells. Insulin regulated this process in part by phosphorylating hnRNPA1, causing it to bind to AU-rich motifs in miRNAs, mediating their secretion into sEVs. Importantly, 43% of insulin-regulated sEV-miRNAs are implicated in obesity and insulin resistance. These include let-7 and miR-103, which we show regulate insulin signaling in AML12 hepatocytes. Together, these findings demonstrate an important layer to insulin\'s regulation of adipose biology and provide a mechanism of tissue crosstalk in obesity and other hyperinsulinemic states.

Aberrant aggregation of misfolded alpha-synuclein (α-syn), a major pathological hallmark of related neurodegenerative diseases such as Parkinson\'s disease (PD), can translocate between cells. Ubiquitin-like 3 (UBL3) is a membrane-anchored ubiquitin-fold protein and post-translational modifier. UBL3 promotes protein sorting into small extracellular vesicles (sEVs) and thereby mediates intercellular communication. Our recent studies have shown that α-syn interacts with UBL3 and that this interaction is downregulated after silencing microsomal glutathione S-transferase 3 (MGST3). However, how MGST3 regulates the interaction of α-syn and UBL3 remains unclear. In the present study, we further explored this by overexpressing MGST3. In the split Gaussia luciferase complementation assay, we found that the interaction between α-syn and UBL3 was upregulated by MGST3. While Western blot and RT-qPCR analyses showed that silencing or overexpression of MGST3 did not significantly alter the expression of α-syn and UBL3, the immunocytochemical staining analysis indicated that MGST3 increased the co-localization of α-syn and UBL3. We suggested roles for the anti-oxidative stress function of MGST3 and found that the effect of MGST3 overexpression on the interaction between α-syn with UBL3 was significantly rescued under excess oxidative stress and promoted intracellular α-syn to extracellular transport. In conclusion, our results demonstrate that MGST3 upregulates the interaction between α-syn with UBL3 and promotes the interaction to translocate intracellular α-syn to the extracellular. Overall, our findings provide new insights and ideas for promoting the modulation of UBL3 as a therapeutic agent for the treatment of synucleinopathy-associated neurodegenerative diseases.