Extracellular vesicles and circulating nucleic acids最新文献

Aim: Mesenchymal stromal cells (MSCs) exert their therapeutic effects in osteoarthritis (OA) primarily through paracrine signaling, including secreted proteins and extracellular vesicle (EV)-associated microRNAs (miRNAs). However, the contribution of tissue origin to the composition and function of these secretomes remains unclear. This study aimed to provide a comprehensive molecular and functional comparison of secretomes from adipose-derived (ASCs), bone marrow-derived MSCs (BMSCs) and human amniotic membrane-derived MSCs, with a specific focus on OA-relevant pathways. Methods: MSCs were immunophenotyped by flow cytometry. Secretomes were profiled for 200 factors and 784 EV-miRNAs. Functional enrichment was performed using Gene Ontology and Reactome databases. In vitro, secretomes were tested on interleukin (IL)-1β-stimulated human chondrocytes to assess modulation of OA-related gene expression. Results: All MSC secretomes shared a core of factors enriched in anti-inflammatory and matrix-regulatory functions. ASCs showed the differential expression of a few modulators, potentially shifting their chondroprotective phenotype. EV-miRNAs further distinguished the MSC types. ASCs and BMSCs clustered closely in both overall miRNA content and functional enrichment, which included pathways for extracellular matrix organization, angiogenesis and IL-6 signaling. BMSC- and ASC-EVs had a higher ratio of OA-protective to destructive miRNAs, including miR-24-3p, miR-125b-5p and miR-222-3p. Functional assays confirmed that all MSC secretomes were effective in suppressing key OA-related genes in inflamed chondrocytes, with ASCs and BMSCs having a stronger activity. Conclusion: These findings support the development of MSC-derived cell-free therapies and emphasize the importance of molecular profiling in MSC source selection. Further studies are warranted to validate these observations and optimize MSC-based interventions for clinical translation in OA.

Aim: Chemotherapy continues to be the frontline treatment for lung cancer patients. However, treatment-related toxicity and off-target effects limit the use of chemotherapy. Therefore, improvements in delivering chemotherapeutics with reduced toxicity to normal tissues are needed. In the present study, we combined nanotechnology with extracellular vesicle (EV) technology to produce tumor-targeted multifunctional EVs (tt-Mfn-EVs) as drug carriers for cancer therapy. Methods: The tt-Mfn-EVs were formulated by exogenously loading EVs with gold nanoparticles conjugated to cisplatin (CDDP) via pH-sensitive coordination ester linkage. Attached to the outer surface of drug-loaded EVs is the transferrin ligand for targeting transferrin receptor (TfR) overexpressing lung cancer cells. Results: The tt-Mfn-EVs were 138.2 nm in size and exhibited greater drug release kinetics at pH 5.5 compared to pH 7.2. They significantly reduced cell viability of A₅₄₉ (TfR high) lung cancer cells compared to HCC₈₂₇ (TfR low) cells and non-targeted EVs. Tt-Mfn-EVs also induced higher levels of apoptosis and DNA damage in A₅₄₉ and HCC₈₂₇ cells compared to control groups. Finally, tt-Mfn-EV-mediated cytotoxicity was minimal in normal human lung fibroblast (MRC-₉) and human embryonic kidney 293 (HEK₂₉₃) cells compared to free CDDP. Conclusion: Our study showed that tt-Mfn-EVs exerted selective and enhanced tumor-targeted cell killing in vitro, providing an opportunity for developing EV-based drug carriers for cancer therapy.

Aim: Small extracellular vesicles (sEVs) are membrane-bound nanoparticles secreted by virtually all cell types that have emerged as promising sources of protein biomarkers for a wide range of diseases, including central nervous system disorders. Blood sampling is the most informative and non-invasive biomarker source. Notably, mouse models represent essential systems for studying in vivo disease mechanisms and testing therapeutic strategies. Therefore, in this study, we investigated the suitability of two different isolation methods for sEV recovery starting from non-terminal mouse blood sampling, with the aim of identifying the most effective protocol for downstream biomarker discovery. Methods: We performed and compared size exclusion chromatography (SEC) and ultracentrifugation followed by iodixanol density gradient (UC-IDG). Additionally, we optimized extracellular vesicle (EV) isolation from small-volume samples of both serum and plasma, since these represent the most used sources for in vivo preclinical biomarker research. Both methods were evaluated in terms of yield, purity, and EV protein content by nanoparticle tracking analysis, electron microscopy, and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) proteomics. Results: SEC showed a higher number of isolated vesicles and EV-associated markers, while reporting a reduced percentage of blood-abundant co-isolated proteins, compared to UC-IDG. The use of plasma as a starting material resulted in a cleaner background, showing fewer protein aggregates. The obtained results emphasize the advantage of SEC in enhancing vesicle yield and purity levels. Conclusion: This work contributes to sEV-derived biomarker research in mouse models by confirming plasma, rather than serum, as the most reliable source of EVs and providing evidence that SEC is more suitable than UC-IDG for EV isolation.

Aim: Representing about 15% of lung cancers, small cell lung cancer (SCLC) is an extremely aggressive disease characterized by rapid growth and early spread, leading to dismal clinical outcomes. In this study, we aimed to investigate the dual roles of exosomal long non-coding RNA (lncRNA) LYPLAL1-DT (LYPLAL1 divergent transcript) in both tumor cells and vascular endothelial cells. Methods: The circulating levels of LYPLAL1-DT were measured using real-time polymerase chain reaction in 13 SCLC patients and 21 normal controls. Exosomes from the supernatant of cell culture medium or serum were extracted through ultracentrifugation and dyed with PKH67 green fluorescent cell linker to identify internalization. Migration and invasion assay, colony formation, Cell Counting Kit-8 (CCK-8), and tube formation assays were used to assess the malignant effects of extracellular RNAs (exRNAs) LYPLAL1-DT in exosomes. Results: Exosomal LYPLAL1-DT is upregulated in SCLC patients and plays a dual role in promoting tumor cell aggressiveness and enhancing pro-angiogenic behavior in endothelial cells, thereby accelerating SCLC progression. Mechanistically, LYPLAL1-DT functions as a competing endogenous RNA, exerting its effects through the miR-204-5p/profilin-2, miR-204-5p/B-cell lymphoma 2 and miR-204-5p/sirtuin 1 regulatory axes. These pathways underscore the pleiotropic effects of exosomal LYPLAL1-DT and underscore its value as a promising therapeutic target. Conclusion: In the current study, we investigated the bidirectional communication mediated by exRNA LYPLAL1-DT between SCLC and endothelial cells, while also exploring its potential regulatory targets. This research provides a potential circulating biomarker for the diagnosis, prognosis, and treatment of SCLC.

Aim: Pig seminal plasma (SP) contains oxytocin (OXT) at levels associated with the fertility outcomes of boars used for artificial insemination (AI). However, OXT easily volatilizes when circulating freely, making it difficult to maintain stability in AI seminal doses. The hypothesis is that OXT is stably carried in seminal extracellular vesicles (EVs). This study aimed to determine the following: (1) whether seminal EVs carry OXT and, if so, where they carry it; (2) the source of seminal EVs carrying OXT; and (3) whether the levels of OXT in seminal EVs are associated with the fertility of AI boars. Methods: Seminal EV samples were isolated by size-exclusion chromatography from entire ejaculates and ejaculate fractions: the first 10 mL of the sperm-rich fraction (SRF; SRF-P1), the remainder of the SRF (SRF-P2), and the post-SRF fraction. The OXT concentration was measured using a direct competitive immunoassay with AlphaLISA technology and an anti-OXT monoclonal antibody. Results: Seminal EVs carry OXT, primarily on the outer surface of the EV membrane, likely within the protein corona. The concentration of OXT in seminal EVs varied among ejaculate fractions (P < 0.0001). It was higher in SRF-P2 (3,017.68 ± 860.52 pg/mL of SP) and post-SRF (3,613.27 ± 935.08 pg/mL of SP) EVs than in SRF-P1 (1,675.15 ± 520.62 pg/mL of SP) EVs. The concentration of OXT in seminal EVs was associated with the fertility outcomes of AI boars. Higher concentrations were found in the seminal EVs of boars with a high farrowing rate and in those with a small litter size. Conclusion: Porcine seminal EVs carry OXT outside of membranes, and those originating from the accessory sex glands are particularly enriched. The association of OXT of seminal EVs with fertility is ambivalent: it enhances farrowing rate while potentially reducing litter size, likely via its effects on myometrial contractility, which facilitates sperm transport but may hinder embryo implantation.

Aim: Small extracellular vesicles (sEVs) are promising noninvasive biomarkers for several malignancies, including thyroid carcinoma (TC). However, their heterogeneity is frequently overlooked in bulk-level analyses. Methods: Plasma samples from TC and healthy controls (HC) were collected for a proximity-dependent barcoding assay (PBA) to identify plasma sEV biomarkers at the single-sEV level. We screened the potential biomarkers using the Panel260 (the panel that detects 260 proteins) of PBA in Cohort 1, and validated them using Panel550 (the panel that detects 550 proteins) in Cohort 2. Results: Plasma exosome counts were significantly elevated in TC compared with those in HC in both Cohort 1 and Cohort 2. Receiver operating characteristic curve analysis showed that sEV counts exhibited an area under the curve > 0.75 in both cohorts. The sEV proteomic analysis revealed that sEV epithelial cell adhesion molecule (EPCAM) levels were significantly increased, whereas claudin-11, integrin alpha X, and lymphocyte-activating 3 were significantly decreased in TC compared with HC. The increase in EPCAM in the plasma and tumor tissues was confirmed by enzyme-linked immunosorbent assay and immunohistochemistry analyses, respectively. The sEV subpopulation analysis further demonstrated that EPCAM⁺ sEVs were significantly elevated in TC compared with HC in both cohorts. The reduction in sEV counts was observed in 18 out of 20 patients after the operation. The decrease in EPCAM⁺ sEVs was observed in 20 patients with TC post-operatively, whereas the reduction in the conventional biomarker serum thyroglobulin (Tg) was observed in 14 patients. TC-derived plasma sEVs promoted TC cell proliferation, migration, invasion, and TC xenograft growth. Conclusion: EPCAM⁺ sEVs could serve as a promising biomarker for the early diagnosis of TC and perform better in monitoring post-operative remission of TC than serum Tg.

Periodontitis is a chronic inflammatory disease characterized by the progressive destruction of both soft (gingiva and periodontal ligament) and hard (cementum and alveolar bone) supporting tissues. The complex periodontal microenvironment often limits the effectiveness of current clinical treatments in achieving functional tissue regeneration. Although mesenchymal and immune cell-based therapies hold promise, concerns related to cell viability and immune compatibility limit their clinical translation. As a natural secretome, small extracellular vesicles (sEVs) are cell-secreted nanoparticles that deliver bioactive molecules for cell-to-cell communication to modulate immune response and promote tissue regeneration. To assess the translational readiness of sEVs therapy, this scoping review first outlines the current clinical trials of mesenchymal stem cells (MSCs)-sEVs in periodontitis, followed by a transition to preclinical application of integrating sEVs with biomaterial scaffolds to enhance localized regenerative outcomes. We then analyzed eight preclinical studies utilizing 3D bioprinted MSCs-sEVs/human umbilical vein endothelial cells-sEVs (or immune cell-derived sEVs) constructs in bone and vasculature regeneration models, and one study related to in vitro periodontal regeneration. These constructs exhibited improved outcomes in osteogenesis, angiogenesis, and immunomodulation, supporting their potential for future translational applications in periodontal therapy. Given the early stage of bioprinted sEVs constructs in periodontitis, we outline critical research gaps and potential future directions to overcome current technical and biological challenges. Together, this review demonstrated the translational trajectory of sEV-based strategies for periodontal regeneration. It offers a potential roadmap for utilizing sEV-based periodontal regeneration across clinical, preclinical, and biofabrication applications, highlighting their potential as next-generation, cell-free therapeutics in regenerative periodontics.

Extracellular vesicles (EVs) are central mediators of intercellular communication in both healthy and malignant states. In normal B lymphocyte (cell) biology, EVs derived from B cells, mast cells, T cells, and mesenchymal stromal cells regulate maturation, antigen presentation, and activation. B cell-derived EVs can either suppress excessive activation to maintain immune homeostasis or amplify responses during an active immune response. Modulation of these responses often occurs via phosphoinositide 3-kinase signaling pathways in recipient cells. In B cell malignancies, such as leukemias, lymphomas, and multiple myeloma, EVs play pivotal roles in disease progression and therapy resistance. Tumor- and stromal-derived EVs can transfer pro-survival proteins, regulatory RNAs, and drug-resistance factors to directly promote tumor progression. In addition, EVs can shape the tumor microenvironment to indirectly promote tumor progression through macrophage polarization, stromal cell reprogramming, and suppression of anti-tumor immunity. Conversely, under certain conditions, B cell EVs can enhance immune surveillance by stimulating T cells and presenting antigen, highlighting their dual potential in cancer biology. Clinically, B cell-derived EVs represent promising liquid biopsy biomarkers: increases in EV abundance, expression of surface antigens, altered protein cargo, and distinct RNA signatures have been associated with disease stage, treatment response, and patient outcomes. Despite this potential, variability in EV isolation and analysis methods remains a barrier to clinical translation. Moving forward, identifying robust biomarker signatures across platforms and clarifying mechanisms of cargo selection and EV uptake will be critical for advancing diagnostic and therapeutic applications. Overall, B cell-derived EVs act as contextual regulators of immune function and malignancy, positioning them as both modulators of disease progression and promising clinical tools.

Although anatomically separate, the gut and lungs are interconnected through intricate pathways involving their respective microbiota, supporting the concept of a gut-lung axis. In the pediatric field, devastating intestinal pathologies such as necrotizing enterocolitis and inflammatory bowel diseases mostly affect preterm infants. In parallel, in the lung, bronchopulmonary dysplasia and chronic obstructive pulmonary disease represent pediatric unmet clinical needs. In this review, we discuss how the extracellular vesicles (EVs), nanoparticles secreted by all cell types, represent a common element in the gut-lung axis. Specifically, EVs play a dual role, serving both as novel disease biomarkers and as promising therapeutic agents.

Aim: Human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) persist in effectively treated HIV-infected individuals, in part due to HIV reservoirs in brain microglia, which express low levels of viral proteins such as Nef. This study aimed to elucidate how microglia release Nef into the extracellular space, where it exerts its biological functions. Methods: Here, we systematically characterized extracellular particles released from immortalized human microglia (h-microglia) expressing Nef alone or after HIV infection. Importantly, we established a novel h-microglia model harboring a stably integrated Nef tagged with green fluorescent protein (Nef.GFP) transgene under an inducible promoter. Extracellular vesicles (EVs) were enriched from culture media and analyzed for morphology, size, concentration and molecular composition, including Nef content, by (super-resolution) fluorescence microscopy, (immunogold) transmission electron microscopy, asymmetric flow field-flow fractionation coupled to a multi-angle light-scattering detector, nanoparticle tracking analysis, and nano-flow cytometry and immunoblotting. Results: Nef.GFP expression increased particle release up to 11.7-fold compared with controls or known stimulants adenosine triphosphate (ATP) and ionomycin. Compared to the latter, the particles were also significantly smaller (root mean square radius, Rrms = 172 nm) and displayed unique protein and density profiles. All data support the EV nature of the released particles. Approximately half of the Nef.GFP-induced EVs contained Nef (45.5% ± 15.8%), with immunogold labeling confirming its intraluminal localization. Notably, infection with HIV isolates NL4-3 and YU-2 likewise produced Nef-positive EVs distinct from virions. Conclusion: Our findings importantly contribute to understanding the source and characteristics of extracellular Nef in the central nervous system of HIV infected individuals and offer new tools to study HIV Nef biology. Nef-laden EVs should be further investigated as potential therapeutic targets in HAND.