Amniotic fluid-derived small extracellular vesicles for predicting postnatal severe outcome of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a life-threatening condition with high morbidity and mortality rates. The survival rate of neonates with severe CDH is reportedly only 10%-15%. However, prenatal prediction of severe cases is difficult, and the discovery of new predictive markers is an urgent issue. In this study, we focused on microRNAs (miRNAs) in amniotic fluid-derived small EVs (AF-sEVs). We identified four miRNAs (hsa-miR-127-3p, hsa-miR-363-3p, hsa-miR-493-5p, and hsa-miR-615-3p) with AUC > 0.8 to classify good prognosis group and poor prognosis group in human study. The AUC for hsa-miR-127-3p and hsa-miR-615-3p, for predicting the poor prognosis, were 0.93 and 0.91, respectively. In addition, in the in vivo study, the miRNA profiles of the lung tissues of CDH rats were different from those of control rats. Additionally, two elevated miRNAs (rno-miR-215-5p and rno-miR-148a-3p) in the lung tissues of CDH rats were increased in the AF-sEVs of CDH rats. Our results suggest that severe CDH neonates can be predicted prenatally with high accuracy using miRNAs contained in AF-sEVs. Furthermore, miRNA profile changes in AF-sEVs reflected the lung status in CDH. Our findings may contribute to the development of advanced perinatal care for patients with CDH.

Extracellular vesicle isolation and counting system (EVics) based on simultaneous tandem tangential flow filtration and large field-of-view light scattering.

Although the isolation and counting of small extracellular vesicles (sEVs) are essential steps in sEV research, an integrated method with scalability and efficiency has not been developed. Here, we present a scalable and ready-to-use extracellular vesicle (EV) isolation and counting system (EVics) that simultaneously allows isolation and counting in one system. This novel system consists of (i) EVi, a simultaneous tandem tangential flow filtration (TFF)-based EV isolation component by applying two different pore-size TFF filters, and (ii) EVc, an EV counting component using light scattering that captures a large field-of-view (FOV). EVi efficiently isolated 50-200 nm-size sEVs from 15 µL to 2 L samples, outperforming the current state-of-the-art devices in purity and speed. EVc with a large FOV efficiently counted isolated sEVs. EVics enabled early observations of sEV secretion in various cell lines and reduced the cost of evaluating the inhibitory effect of sEV inhibitors by 20-fold. Using EVics, sEVs concentrations and sEV PD-L1 were monitored in a 23-day cancer mouse model, and 160 clinical samples were prepared and successfully applied to diagnosis. These results demonstrate that EVics could become an innovative system for novel findings in basic and applied studies in sEV research.

Small Extracellular Vesicle-Derived Circular RNA hsa_circ_0007386 as a Biomarker for the Diagnosis of Pleural Mesothelioma.

Pleural mesothelioma (PM) is a highly aggressive tumor that is caused by asbestos exposure and lacks effective therapeutic regimens. Current procedures for PM diagnosis are invasive and can take a long time to reach a definitive result. Small extracellular vesicles (sEVs) have been identified as important communicators between tumor cells and their microenvironment via their cargo including circular RNAs (circRNAs). CircRNAs are thermodynamically stable, highly conserved, and have been found to be dysregulated in cancer. This study aimed to identify potential biomarkers for PM diagnosis by investigating the expression of specific circRNA gene pattern (hsa_circ_0007386) in cells and sEVs using digital polymerase chain reaction (dPCR). For this reason, 5 PM, 14 non-PM, and one normal mesothelial cell line were cultured. The sEV was isolated from the cells using the gold standard ultracentrifuge method. The RNA was extracted from both cells and sEVs, cDNA was synthesized, and dPCR was run. Results showed that hsa_circ_0007386 was significantly overexpressed in PM cell lines and sEVs compared to non-PM and normal mesothelial cell lines (p < 0.0001). The upregulation of hsa_circ_0007386 in PM highlights its potential as a diagnostic biomarker. This study underscores the importance and potential of circRNAs and sEVs as cancer diagnostic tools.

Small extracellular vesicle CA1 as a promising diagnostic biomarker for nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC), a malignant cancer originating from the epithelial cells of the nasopharynx, presents diagnostic challenges with current methods such as plasma Epstein-Barr virus (EBV) DNA testing showing limited efficacy. This study focused on identifying small extracellular vesicle (sEV) proteins as potential noninvasive biomarkers to enhance NPC diagnostic accuracy. We isolated sEVs from plasma and utilized 4D label-free proteomics to identify differentially expressed proteins (DEPs) among healthy controls (NC = 10), early-stage NPC (E-NPC = 10), and late-stage NPC (L-NPC = 10). Eighteen sEV proteins were identified as potential biomarkers. Subsequently, parallel reaction monitoring (PRM) proteomic analysis preliminarily confirmed sEV carbonic anhydrase 1 (CA1) as a highly promising biomarker for NPC, particularly in early-stage diagnosis (NC = 15; E-NPC = 10; L-NPC = 15). To facilitate this, we developed an automated, high-throughput and highly sensitive CA1 immune-chemiluminescence chip technology characterized by a broad linear detection range and robust controls. Further validation in an independent retrospective cohort (NC = 89; E-NPC = 39; L-NPC = 172) using this technology confirmed sEV CA1 as a reliable diagnostic biomarker for NPC (AUC = 0.9809) and E-NPC (AUC = 0.9893), independent of EBV-DNA testing. Notably, sEV CA1 exhibited superior diagnostic performance compared to EBV-DNA, with a significant incremental net reclassification improvement of 27.61 % for NPC and 72.11 % for E-NPC detection. Thus, this study identifies sEV CA1 as an innovative diagnostic biomarker for NPC and E-NPC independent of EBV-DNA. Additionally, it establishes an immune-chemiluminescence chip technology for the detection of sEV CA1 protein, paving the way for further validation and clinical application.

Enhanced immunosuppressive capability of mesenchymal stem cell-derived small extracellular vesicles with high expression of CD73 in experimental autoimmune uveitis.

BACKGROUND: Autoimmune uveitis is an inflammatory disease triggered by an aberrant immune response. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) are emerging as potential therapeutic agents for this condition. CD73, an ectoenzyme present on MSC-sEVs, is involved in mitigating inflammation by converting extracellular adenosine monophosphate into adenosine. We hypothesize that the inhibitory effect of MSC-sEVs on experimental autoimmune uveitis (EAU) could be partially attributed to the surface expression of CD73. METHODS: To investigate novel therapeutic approaches for autoimmune uveitis, we performed lentiviral transduction to overexpress CD73 on the surface of MSC-sEVs, yielding CD73-enriched MSC-sEVs (sEVs-CD73). Mice with interphotoreceptor retinoid-binding protein (IRBP)-induced EAU were grouped randomly and treated with 50 µg MSC-sEVs, vector infected MSC-sEVs, sEVs-CD73 or PBS via single tail vein injection. We evaluated the clinical and histological features of the induced mice and analyzed the proportion and functional capabilities of T helper cells. Furthermore, T-cells were co-cultured with various MSC-sEVs in vitro, and we quantified the resulting inflammatory response to assess the potential therapeutic benefits of sEVs-CD73. RESULTS: Compared to MSC-sEVs, sEVs-CD73 significantly alleviates EAU, leading to reduced inflammation and diminished tissue damage. Treatment with sEVs-CD73 results in a decreased proportion of Th1 cells in the spleen, draining lymph nodes, and eyes, accompanied by an increased proportion of regulatory T-cells (Treg cells). In vitro assays further reveal that sEVs-CD73 inhibits T-cell proliferation, suppresses Th1 cells differentiation, and enhances Treg cells proportion. CONCLUSION: Over-expression of CD73 on MSC-sEVs enhances their immunosuppressive effects in EAU, indicating that sEVs-CD73 has the potential as an efficient immunotherapeutic agent for autoimmune uveitis.

Appearance of small extracellular vesicles in the mouse pregnant serum and the localization in placentas.

Exosomes or small extracellular vesicles (sEVs) are present in the blood of pregnant mice and considered to be involved in pregnancy physiology. Although sEVs in pregnant periods are proposed to be derived from placentas, sEVs-producing cells are not well known in mouse placentas. We studied the dynamics and localization of sEVs in pregnant serum and placentas, and examined gestational variation of microRNA (miRNA). Serums and placentas were collected from non-pregnant (NP) and pregnant mice throughout the entire gestational day (Gd). EVs were purified from serums and total RNA was isolated from EVs. Nanoparticle-tracking assay (NTA) revealed that the rates of sEVs in EVs are 53% at NP, and increased to 80.1% at Gd 14.5 and 97.5% at Gd 18.5. Western blotting on EVs showed positive reactivity to the tetraspanin markers and clarified that the results using anti-CD63 antibody were most consistent with the sEVs appearance detected by NTA. Serum EVs also showed a positive reaction to the syncytiotrophoblast marker, syncytin-1. Immunohistostaining using anti-CD63 antibody showed positive reactions in mouse placentas at the syncytiotrophoblasts and endothelial cells of the fetal capillaries. Quantitative PCR revealed that significantly higher amounts of miRNAs were included in the sEVs of Gd 18.5. Our results suggested that sEVs are produced in the mouse placenta and transferred to maternal or fetal bloodstreams. sEVs are expected to have a miRNA-mediated physiological effect and become useful biomarkers reflecting the pregnancy status.

MiR-4465-modified mesenchymal stem cell-derived small extracellular vesicles inhibit liver fibrosis development via targeting LOXL2 expression. / MiR-4465修饰的间质干细胞来源的小细胞外囊泡通过靶向LOXL2表达抑制肝纤维化的进展.

Liver fibrosis is a significant health burden, marked by the consistent deposition of collagen. Unfortunately, the currently available treatment approaches for this condition are far from optimal. Lysyl oxidase-like protein 2 (LOXL2) secreted by hepatic stellate cells (HSCs) is a crucial player in the cross-linking of matrix collagen and is a significant target for treating liver fibrosis. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) have been proposed as a potential treatment option for chronic liver disorders. Previous studies have found that MSC-sEV can be used for microRNA delivery into target cells or tissues. It is currently unclear whether microRNA-4465 (miR-4465) can target LOXL2 and inhibit HSC activation. Additionally, it is uncertain whether MSC-sEV can be utilized as a gene therapy vector to carry miR-4465 and effectively inhibit the progression of liver fibrosis. This study explored the effect of miR-4465-modified MSC-sEV (MSC-sEVmiR-4465) on LOXL2 expression and liver fibrosis development. The results showed that miR-4465 can bind specifically to the promoter of the LOXL2 gene in HSC. Moreover, MSC-sEVmiR-4465 inhibited HSC activation and collagen expression by downregulating LOXL2 expression in vitro. MSC-sEVmiR-4465 injection could reduce HSC activation and collagen deposition in the CCl4-induced mouse model. MSC-sEVmiR-4465 mediating via LOXL2 also hindered the migration and invasion of HepG2 cells. In conclusion, we found that MSC-sEV can deliver miR-4465 into HSC to alleviate liver fibrosis via altering LOXL2, which might provide a promising therapeutic strategy for liver diseases.

Multiarmed DNA jumper and metal-organic frameworks-functionalized paper-based bioplatform for small extracellular vesicle-derived miRNAs assay.

Small extracellular vesicle-derived microRNAs (sEV-miRNAs) have emerged as promising noninvasive biomarkers for early cancer diagnosis. Herein, we developed a molecular probe based on three-dimensional (3D) multiarmed DNA tetrahedral jumpers (mDNA-Js)-assisted DNAzyme activated by Na+, combined with a disposable paper-based electrode modified with a Zr-MOF-rGO-Au NP nanocomplex (ZrGA) to fabricate a novel biosensor for sEV-miRNAs Assay. Zr-MOF tightly wrapped by rGO was prepared via a one-step method, and it effectively aids electron transfer and maximizes the effective reaction area. In addition, the mechanically rigid, and nanoscale-addressable mDNA-Js assembled from the bottom up ensure the distance and orientation between fixed biological probes as well as avoid probe entanglement, considerably improving the efficiency of molecular hybridization. The fabricated bioplatform achieved the sensitive detection of sEV-miR-21 with a detection limit of 34.6 aM and a dynamic range from100 aM to 0.2 µM. In clinical blood sample tests, the proposed bioplatform showed results highly consistent with those of qRT-PCRs and the signal increased proportionally with the NSCLC staging. The proposed biosensor with a portable wireless USB-type analyzer is promising for the fast, easy, low-cost, and highly sensitive detection of various nucleic acids and their mutation derivatives, making it ideal for POC biosensing.

Small extracellular vesicles of organoid-derived human retinal stem cells remodel Müller cell fate via miRNA: A novel remedy for retinal degeneration.

Remodeling retinal Müller glial fate, including gliosis inhibition and pro-reprogramming, represents a crucial avenue for treating degenerative retinal diseases. Stem cell transplantation exerts effects on modulating retinal Müller glial fate. However, the optimized stem cell products and the underlying therapeutic mechanisms need to be investigated. In the present study, we found that retinal progenitor cells from human embryonic stem cell-derived retinal organoids (hERO-RPCs) transferred extracellular vesicles (EVs) into Müller cells following subretinal transplantation into RCS rats. Small EVs from hERO-RPCs (hERO-RPC-sEVs) were collected and were found to delay photoreceptor degeneration and protect retinal function in RCS rats. hERO-RPC-sEVs were taken up by Müller cells both in vivo and in vitro, and inhibited gliosis while promoting early dedifferentiation of Müller cells. We further explored the miRNA profiles of hERO-RPC-sEVs, which suggested a functional signature associated with neuroprotection and development, as well as the regulation of stem cell and glial fate. Mechanistically, hERO-RPC-sEVs might regulate the fate of Müller cells by miRNA-mediated nuclear factor I transcription factors B (NFIB) downregulation. Collectively, our findings offer novel mechanistic insights into stem cell therapy and promote the development of EV-centered therapeutic strategies.

Small extracellular vesicles derived from lipopolysaccharide-preconditioned dental follicle cells inhibit cell apoptosis and alveolar bone loss in periodontitis.

OBJECTIVE: This study aimed to explore the effects of small extracellular vesicles derived from lipopolysaccharide-preconditioned dental follicle cells (L-D-sEV) on periodontal ligament cells from periodontitis affected teeth (p-PDLCs) in vitro and experimental periodontitis in mice. DESIGN: In vitro, the biological function of p-PDLCs and the underlying molecular mechanism were investigated by flow cytometry, Western blot, and quantitative real-time PCR (qRT-PCR) analysis. Eighteen-eight-week-old male C57BL/6 mice were randomly divided into three groups: control (Con), periodontitis (Peri), and L-D-sEV groups. Mice periodontitis model was induced by placing the 5-0 silk thread (around the maxillary second molar) and P.gingivalis (1 ×107 CFUs per mouse). In vivo, the alveolar bone loss, osteoclast activity, and macrophage polarization were measured by micro-computed tomography and histological analysis. RESULTS: In vitro, the RANKL/OPG ratio and phosphorylation of JNK and P38 protein levels of p-PDLCs were significantly decreased after L-D-sEV administration. Besides, flow cytometry and qRT-PCR analysis showed that L-D-sEV reduced apoptosis of p-PDLCs, down-regulated apoptosis-related genes Caspase-3 and BCL-2-Associated X expression, and up-regulated B-cell lymphoma-2 gene levels. In vivo, L-D-sEV administration significantly reduced alveolar bone loss, inhibited osteoclast activity, and induced M2 polarization. The histological analysis showed that iNOS/CD206, RANKL/OPG, p-JNK/JNK, and p-P38/P38 ratios were significantly lower in the L-D-sEV group than in the Peri group. CONCLUSIONS: L-D-sEV administration alleviated alveolar bone loss by mediating RANKL/OPG-related osteoclast activity and M2 macrophage polarization, alleviating p-PDLCs apoptosis and proliferation via the JNK and P38 pathways.

Effective treatment of optic neuropathies by intraocular delivery of MSC-sEVs through augmenting the G-CSF-macrophage pathway.

Optic neuropathies, characterized by injury of retinal ganglion cell (RGC) axons of the optic nerve, cause incurable blindness worldwide. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) represent a promising "cell-free" therapy for regenerative medicine; however, the therapeutic effect on neural restoration fluctuates, and the underlying mechanism is poorly understood. Here, we illustrated that intraocular administration of MSC-sEVs promoted both RGC survival and axon regeneration in an optic nerve crush mouse model. Mechanistically, MSC-sEVs primarily targeted retinal mural cells to release high levels of colony-stimulating factor 3 (G-CSF) that recruited a neural restorative population of Ly6Clow monocytes/monocyte-derived macrophages (Mo/MΦ). Intravitreal administration of G-CSF, a clinically proven agent for treating neutropenia, or donor Ly6Clow Mo/MΦ markedly improved neurological outcomes in vivo. Together, our data define a unique mechanism of MSC-sEV-induced G-CSF-to-Ly6Clow Mo/MΦ signaling in repairing optic nerve injury and highlight local delivery of MSC-sEVs, G-CSF, and Ly6Clow Mo/MΦ as therapeutic paradigms for the treatment of optic neuropathies.

Tumor-derived small extracellular vesicles facilitate omental metastasis of ovarian cancer by triggering activation of mesenchymal stem cells.

BACKGROUND: Omental metastasis is the major cause of ovarian cancer recurrence and shortens patient survival, which can be largely attributed to the dynamic evolution of the fertile metastatic microenvironment driven by cancer cells. Previously, we found that adipose-derived mesenchymal stem cells (ADSCs) undergoing a phenotype shift toward cancer-associated fibroblasts (CAFs) participated in the orchestrated omental premetastatic niche for ovarian cancer. Here, we aim to elucidate the underlying mechanisms. METHODS: Small extracellular vesicles were isolated from ovarian cancer cell lines (ES-2 and its highly metastatic subline, ES-2-HM) and patient ascites using ultracentrifugation. Functional experiments, including Transwell and EdU assays, and molecular detection, including Western blot, immunofluorescence, and RT-qPCR, were performed to investigate the activation of ADSCs in vitro. High-throughput transcriptional sequencing and functional assays were employed to identify the crucial functional molecules inducing CAF-like activation of ADSCs and the downstream effector of miR-320a. The impact of extracellular vesicles and miR-320a-activated ADSCs on tumor growth and metastasis was assessed in subcutaneous and orthotopic ovarian cancer xenograft mouse models. The expression of miR-320a in human samples was evaluated using in situ hybridization staining. RESULTS: Primary human ADSCs cocultured with small extracellular vesicles, especially those derived from ES-2-HM, exhibited boosted migration, invasion, and proliferation capacities and elevated α-SMA and FAP levels. Tumor-derived small extracellular vesicles increased α-SMA-positive stromal cells, fostered omental metastasis, and shortened the survival of mice harboring orthotopic ovarian cancer xenografts. miR-320a was abundant in highly metastatic cell-derived extracellular vesicles, evoked dramatic CAF-like transition of ADSCs, targeted the 3'-untranslated region of integrin subunit alpha 7 and attenuated its expression. miR-320a overexpression in ovarian cancer was associated with omental metastasis and shorter survival. miR-320a-activated ADSCs facilitated tumor cell growth and omental metastasis. Depletion of integrin alpha 7 triggered CAF-like activation of ADSCs in vitro. Video Abstract CONCLUSIONS: miR-320a in small extracellular vesicles secreted by tumor cells targets integrin subunit alpha 7 in ADSCs and drives CAF-like activation, which in turn facilitates omental metastasis of ovarian cancer.

Dynamic expression analysis of peripheral blood derived small extracellular vesicle miRNAs in sepsis progression.

Immune disorders caused by sepsis have recently drawn much attention. We sought to dynamically monitor the expression of small extracellular vesicle (sEV) miRNAs in peripheral blood during sepsis to explore these miRNAs as potential biomarkers for monitoring immune function in sepsis patients. This study included patients with sepsis. Blood samples were obtained from 10 patients on the first through 10th days, the 12th day and the 14th day since sepsis onset, resulting in 120 collected samples. Serum sEVs were extracted from peripheral venous blood, and levels of MIR497HG, miR-195, miR-497, and PD-L1 in serum sEVs were detected by qPCR, and clinical information was recorded. Our study revealed that the levels of MIR497HG, miR-195, miR-497 and PD-L1 in serum sEVs showed periodic changes; the time from peak to trough was approximately 4-5 days. The levels of sEV MIR497HG and miR-195 had a positive linear relationship with SOFA score (r values were -0.181 and -0.189; p values were 0.048 and 0.039, respectively). The recorded quantities of sEV MIR497HG, miR-195 and PD-L1 showed a substantial correlation with ARDS. ROC curve analysis revealed that sEV MIR497HG, miR-195 and miR-497 could predict the 28-day mortality of sepsis patients with an AUC of 0.66, 0.68 and 0.72, respectively. Levels of sEVs MIR497HG, miR-195, miR-497 and PD-L1 showed periodic changes with the immune status of sepsis, which provides a new exploration direction for immune function biomarkers and immunotherapy timing in sepsis patients.

Polarized Desmosome and Hemidesmosome Shedding via Small Extracellular Vesicles is an Early Indicator of Outer Blood-Retina Barrier Dysfunction.

The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal-side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases (e.g., AMD).

Circulating plasma miR-23b-3p as a biomarker target for idiopathic Parkinson's disease: comparison with small extracellular vesicle miRNA.

Background: Parkinson's disease (PD) is an increasingly common neurodegenerative condition, which causes movement dysfunction and a broad range of non-motor symptoms. There is no molecular or biochemical diagnosis test for PD. The miRNAs are a class of small non-coding RNAs and are extensively studied owing to their altered expression in pathological states and facile harvesting and analysis techniques. Methods: A total of 48 samples (16 each of PD, aged-matched, and young controls) were recruited. The small extracellular vesicles (sEVs) were isolated and validated using Western blot, transmission electron microscope, and nanoparticle tracking analysis. Small RNA isolation, library preparation, and small RNA sequencing followed by differential expression and targeted prediction of miRNA were performed. The real-time PCR was performed with the targeted miRNA on PD, age-matched, and young healthy control of plasma and plasma-derived sEVs to demonstrate their potential as a diagnostic biomarker. Results: In RNA sequencing, we identified 14.89% upregulated (fold change 1.11 to 11.04, p < 0.05) and 16.54% downregulated (fold change -1.04 to -7.28, p < 0.05) miRNAs in PD and controls. Four differentially expressed miRNAs (miR-23b-3p, miR-29a-3p, miR-19b-3p, and miR-150-3p) were selected. The expression of miR-23b-3p was "upregulated" (p = 0.002) in plasma, whereas "downregulated" (p = 0.0284) in plasma-derived sEVs in PD than age-matched controls. The ROC analysis of miR-23b-3p revealed better AUC values in plasma (AUC = 0.8086, p = 0.0029) and plasma-derived sEVs (AUC = 0.7278, p = 0.0483) of PD and age-matched controls. Conclusion: We observed an opposite expression profile of miR-23b-3p in PD and age-matched healthy control in plasma and plasma-derived sEV fractions, where the expression of miR-23b-3p is increased in PD plasma while decreased in plasma-derived sEV fractions. We further observed the different miR-23b-3p expression profiles in young and age-matched healthy control.

VAMP5 and distinct sets of cognate Q-SNAREs mediate exosome release.

Small extracellular vesicles (sEVs) are largely classified into two types, plasma-membrane derived sEVs and endomembrane-derived sEVs. The latter type (referred to as exosomes herein) is originated from late endosomes or multivesicular bodies (MVBs). In order to release exosomes extracellularly, MVBs must fuse with the plasma membrane, not with lysosomes. In contrast to the mechanism responsible for MVB-lysosome fusion, the mechanism underlying the MVB-plasma membrane fusion is poorly understood. Here, we systematically analyze soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family proteins and identify VAMP5 as an MVB-localized SNARE protein required for exosome release. Depletion of VAMP5 in HeLa cells impairs exosome release. Mechanistically, VAMP5 mediates exosome release by interacting with SNAP47 and plasma membrane SNARE Syntaxin 1 (STX1) or STX4 to release exosomes. VAMP5 is also found to mediate asymmetric exosome release from polarized Madin-Darby canine kidney (MDCK) epithelial cells through interaction with the distinct sets of Q-SNAREs, suggesting that VAMP5 is a general exosome regulator in both polarized cells and non-polarized cells.Key words: exosome, small extracellular vesicle (sEV), multivesicular body, SNARE, VAMP5.

Different Biofluids, Small Extracellular Vesicles or Exosomes: Structural Analysis in Atherosclerotic Cardiovascular Disease Using Electron Microscopy Techniques.

Small extracellular vesicles (sEVs) or exosomes are secretory vesicles largely involved in cell-cell communications and found to play a role in development as well as diseases including atherosclerosis. They hold a huge potential for translational research by devising better clinical diagnostics, biomarker discovery, drug delivery, and therapeutic strategies. Variations terms of morphology and distribution are crucial to biological function integrity. Moreover, it is dependent on susceptibility to influential factors of the environment like cell stress, inflammation, and secretion by different cells in subsequent biofluids. We have observed the morphological variations in sEVs or exosomes freshly isolated from patients with atherosclerotic cardiovascular disease (AsCVD), in blood plasma, saliva, and urine biofluids compared to healthy controls. High-resolution images were obtained by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) for the characterization of sEVs morphology. Western blotting and immuno-TEM gold labeling confirmed the presence of exosome markers. For the first time, we report size and shape variations, which suggest the existence of different functions of sEVs in the disease state. Morphological variations in sEVs were observed significantly in noninvasive AsCVD saliva and urine samples, important to understand the cell behavior and physiological state. These variations will be useful to investigate their possible role in the disease process.

Effects of sEV derived from SHED and DPSC on the proliferation, migration and osteogenesis of PDLSC.

Introduction: Periodontitis is a highly prevalent oral disease characterized by irreversible bone resorption and tooth loss. The proliferation, migration and osteogenic differentiation of periodontal ligament stem cell (PDLSC) are crucial to the regeneration of periodontal bone defects. There is increasing evidence that small extracellular vesicle (sEV) derived from pulp stem cell, including human exfoliated deciduous teeth stem cell (SHED) and human dental pulp stem cell (DPSC), is a potential mediator for bone tissue regeneration. However, which one is more suitable for periodontal bone formation still remains to be studied. Methods: In this study, NTA and BCA were performed to compare the productivity of sEV derived from SHED (SHED-sEV) and sEV derived from DPSC (DPSC-sEV). CCK-8, transwell assay, alkaline phosphatase staining and activity assay, alizarin red staining, qRT-PCR, and western blotting were conducted to detect the proliferation, migration, and osteogenesis of PDLSCs coculturing with SHED-sEV or DPSC-sEV. Results: The secretory efficiency of SHED-sEV was much higher than that of DPSC-sEV. The cellular uptake of sEVs could promote the proliferation, migration and osteogenesis of DPLSCs. Compared with DPSC-sEV, SHED-sEV showed better ability in such promotion. Conclusions: SHED-sEV showed higher productivity and better osteogenic induction ability than DPSC-sEV. Thus, SHED-sEV may be a more promising candidate for periodontal bone regeneration.

Identifying Serum Small Extracellular Vesicle MicroRNA as a Noninvasive Diagnostic and Prognostic Biomarker for Ovarian Cancer.

There remains a lack of effective and noninvasive methods for the diagnosis and prognosis prediction of epithelial ovarian carcinoma (EOC). Here, we investigated the possibility of serum-derived small extracellular vesicle (sEV) microRNAs (miRNAs) as potential biomarkers for distinguishing between benign and malignant adnexal masses and predicting the prognosis of EOC patients. A serum sEV miRNA model for identifying the EOC (sEVmiR-EOC) was successfully established in the training cohort. Furthermore, the sEVmiR-EOC model was confirmed in the testing cohort and validation cohort, demonstrating robust diagnostic accuracy. The sEVmiR-EOC model showed better performance than carbohydrate antigen 125 (CA125) in discriminating patients with stage I EOC from benign patients. Using EOC samples and follow-up data, we identified miR-141-3p and miR-200c-3p as potential prognostic predictors. Finally, we confirmed the change of the sEVmiR-EOC RiskScore between the preoperative and postoperative samples and found that the sEVmiR-EOC model could predict the prognosis of EOC patients.

FUS-mediated HypEVs: Neuroprotective effects against ischemic stroke.

Few studies have investigated the properties and protein composition of small extracellular vesicles (sEVs) derived from neurons under hypoxic conditions. Presently, the extent of the involvement of these plentiful sEVs in the onset and progression of ischemic stroke remains an unresolved question. Our study systematically identified the characteristics of sEVs derived from neurons under hypoxic conditions (HypEVs) by physical characterization, sEV absorption, proteomics and transcriptomics analysis. The effects of HypEVs on neurites, cell survival, and neuron structure were assessed in vitro and in vivo by neural complexity tests, magnetic resonance imaging (MRI), Golgi staining, and Western blotting of synaptic plasticity-related proteins and apoptotic proteins. Knockdown of Fused in Sarcoma (FUS) small interfering RNA (siRNA) was used to validate FUS-mediated HypEV neuroprotection and mitochondrial mRNA release. Hypoxia promoted the secretion of sEVs, and HypEVs were more easily taken up and utilized by recipient cells. The MRI results illustrated that the cerebral infarction volume was reduced by 45% with the application of HypEVs, in comparison to the non- HypEV treatment group. Mechanistically, the FUS protein is necessary for the uptake and neuroprotection of HypEVs against ischemic stroke as well as carrying a large amount of mitochondrial mRNA in HypEVs. However, FUS knockdown attenuated the neuroprotective rescue capabilities of HypEVs. Our comprehensive dataset clearly illustrates that FUS-mediated HypEVs deliver exceptional neuroprotective effects against ischemic stroke, primarily through the maintenance of neurite integrity and the reduction of mitochondria-associated apoptosis.