Journal of extracellular biology最新文献

Extracellular vesicles (EVs) are nanosized, membrane-enclosed particles released by cells, facilitating intercellular communication via the transfer of bioactive molecules from producing to recipient cells. EVs are involved in several physiological and pathological processes, including cancer progression. The glycosylation of EVs influences the attachment and uptake by recipient cells, among other processes. In this study, we investigated the glycan profile of EVs from canine osteosarcoma (OS) cells and non-cancerous canine osteoblasts in vitro, using lectin blots, following EV separation with ultracentrifugation (UC), size exclusion chromatography (SEC), and a commercial precipitation kit (TEI). Beyond phenotypic differences, purity and yield, separation methods led to heterogeneous protein glycosylation patterns of EV preparations, with the least reproducibility in TEI preparations. In cellular comparison, SEC revealed contrasting results, most consistently showing higher levels of specific sugars in EVs from canine osteoblasts, including the detection of a unique glycoprotein absent in UC and TEI preparations. Osteosarcoma cells and their EVs exhibited a relative reduction of glycoproteins compared to osteoblasts and their EVs. Additionally, specific sugars were more abundant in EV preparations relative to their corresponding cell lysates. In conclusion, this study highlights the critical influence of separation methods on EV characteristics, leading to differing glycan patterns.

The interaction between mutated leucine-rich repeat kinase 2 (LRRK2) and the death adaptor protein FADD accounts for apoptotic death of dopaminergic neurons in familial Parkinson's disease (PD) driven by LRRK2 mutations. Disrupting this pathogenic interaction using constrained peptides is a promising therapeutic strategy to mitigate apoptotic neuronal death in PD. However, efficiently delivering these therapeutic peptides to disease-relevant cells within the central nervous system (CNS) remains challenging due to degradation in circulation and poor blood-brain barrier and cell membrane penetration. Here, we present a strategy to use extracellular vesicles (EVs) as delivery vehicles for the therapeutic peptides to enhance their cellular uptake and CNS targeting. Following an optimized passive loading approach, we successfully packaged these peptides into EVs, improving their cellular uptake by disease-relevant neural cells in vitro and brain biodistribution in mice following intravenous administration. EV-based delivery enhanced the therapeutic efficacy of these peptides in disrupting FADD-LRRK2 interactions, reducing downstream caspase signaling and neuronal death in cellular models of PD compared to the free peptide format. These findings support the use of EVs as a promising shuttle for peptide-based therapies in PD and potentially other neurological disorders.

Extracellular vesicles (EVs) released from tissues can be found in human biofluids. EVs reflect the phenotypic state of their cell of origin, carrying informative molecular biomarkers within and between tissues, and represent a promising target for liquid biopsy. However, the heterogeneity of EVs in their surface composition, luminal content, biogenesis and cellular origin poses a challenge for selective enrichment and validation of origin-specific EVs from the complex pool of circulating EVs. Another obstacle for translating EVs into liquid biopsy applications is the wide variety of separation and characterization methods, many of which lack standardization and reproducibility. In this review, we summarize current knowledge on tissue-specific EVs, highlighting their potential as indicators of tissue health and disease, as well as the existing challenges and limitations. From the existing literature, we identify a compelling need for better validation and reproducibility studies to support the development of tissue-specific EV applications. Identifying reliable tissue-enriched biomarkers, in particular, will be required to enable further insights into the physiology and pathology of EV source tissues. We also propose some considerations to address future guidelines on the topic. Together, these approaches will help to establish EV liquid biopsy applications as a keystone of translational medicine.

Pediatric high-grade gliomas (pHGGs) account for 20% of childhood brain tumours and are associated with poor survival. Currently, pHGG detection relies on MRI, a costly and time-consuming procedure. Extracellular vesicles (EVs) carry molecular markers indicative of their cellular origin and can be isolated from various biofluids, offering an alternative approach. Recent studies showed that pHGGs contain cells that molecularly and morphologically resemble radial glia (RG), a type of neural progenitor. Given that RGs are normally exclusive to the developing brain, we hypothesized that EVs secreted from RG-like glioma cells serve as a pHGG biomarker. However, there are no established molecular markers to specifically detect RG-derived EVs. To address this, we first identified a combination of surface markers to differentiate EVs derived from RG-like glioma cells from those of non-RG cells. We next validated the expression of these markers in patient-derived cell lines. Analysis of EVs isolated from cell culture media confirmed the presence of these markers, along with other tumour-associated markers including targets of chimeric antigen receptor (CAR) T cell therapy. Taken together, our results showed that RG-derived EVs can be isolated and detected by the combination of markers, laying a groundwork for developing a novel EV-based biomarker for pHGGs.

Extra-large extracellular vesicles (XLEVs), with diameters > 600 nm, are increasingly recognised as mediators of specialized modes of intercellular communication; however, the molecular mechanisms governing their biogenesis and functional regulation remain poorly understood. Here, we show that PI3K–Rab18-GDP signalling promotes the secretion of XLEVs from human mesenchymal stem cells (hMSCs) and fibroblasts. These vesicles are highly enriched in sonic hedgehog (SHH) and display potent pro-angiogenic activity. We further demonstrate that Rab18 functions as a key regulator of this pathway specifically in its GDP-bound form, which can be enriched by the Rab inhibitor CID1067700 or by pharmacological activation of PI3K using SF1670. Rab18-GDP preferentially accumulates in the perinuclear region, where it promotes the formation of SHH-XLEV precursors from endosomal compartments. Mechanistically, PI3K–Rab18-GDP signalling recruits heat shock protein 90α (Hsp90α) and neutral sphingomyelinase 2 (nSMase2), facilitating polarized release of SHH-XLEVs from the perinuclear–plasma membrane interface, accompanied by an Hsp90α-enriched extracellular assembly. Together, these findings identify a PI3K–Rab18-GDP–dependent secretory pathway for SHH-XLEVs and provide a framework for understanding how XLEV biogenesis is coupled to SHH-associated angiogenic signalling in developmental and regenerative contexts.

Extracellular vesicles (EVs) are key mediators of maternal–foetal communication, regulating placental function and foetal development through the transfer of bioactive molecules. Although placental EVs play a crucial role in placental function during pregnancy, their contribution to foetal development, notably foetal brain, remains poorly understood. Human cytomegalovirus (HCMV) is the most common virus transmitted in utero and a leading cause of infectious brain malformations. Although certain central nervous system lesions caused by HCMV are explained, the neuropathogenesis of congenital infection remains poorly understood. In this study, we demonstrate that EVs from healthy placentas promote neurogenesis. However, EVs from HCMV-infected placentas lose this neurogenic potential, impairing differentiation and migration of neural stem cells, perturbations that may contribute to the neurodevelopmental defects observed in congenital HCMV infections. miRNA profiling revealed profound infection-induced changes, including the incorporation of viral miRNAs and dysregulation of host miRNAs involved in neurogenesis. These findings highlight the critical role of placental EVs in foetal brain development and their contribution to HCMV neuropathogenesis.

Human milk extracellular vesicles (HMEVs), secreted by mammary epithelial cells, are enriched in bioactive molecules that support intestinal epithelial integrity. Among these, oxylipins, that is, lipid mediators derived from polyunsaturated fatty acids, are gaining interest for their immunomodulatory and neuroprotective functions in breastfed infants. However, current workflows for oxylipin profiling in HMEVs often lack sensitivity or breadth, limiting mechanistic insights. This study presents an optimised workflow for comprehensive oxylipin profiling in HMEVs. HMEVs were isolated via size-exclusion chromatography and ultracentrifugation, followed by characterisation using attenuated total reflectance–Fourier transform infrared spectroscopy, Western blotting, Exoview immunocapture, tunable resistive pulse sensing and transmission electron microscopy. The influence of different pre-analytical protocols on HMEV recovery was assessed. Cryolysis with liquid nitrogen was employed for vesicle lysis before targeted oxylipin quantification using ultra-performance liquid chromatography–tandem mass spectrometry. The analysis of 10 human milk samples revealed 9,10-DiHOME, 12,13-DiHOME and 11,12-EET as the most abundant oxylipins, with concentrations ranging from 0.5 to 3.7, 0.8 to 4.5 and 0.1 to 0.3 nM, respectively. This refined pipeline enables in-depth oxylipin profiling in HMEVs and serves as a robust platform for future in vitro and in vivo investigations into EV-mediated lipid signalling.

Regenerative vascular medicine research has positioned mesenchymal stromal cells (MSCs) as a leading candidate to treat ischemic diseases. Recent studies have highlighted the emerging role of small extracellular vesicles (sEVs) produced by MSCs in their own potential. This study explores a strategy to improve the angiogenic potential of MSCs through the acquisition of endothelial features. Umbilical cord Wharton's jelly MSCs were cultured in fetal bovine serum-free endothelial growth medium under hypoxic conditions (SH-MSCs). sEVs were characterised by a multimodal approach: visualisation, count and particle size distribution, sEVs surface antigen, proangiogenic potential and ability to internalise into recipient cells. Compared with MSCs, SH-MSCs exhibited significant morphological and phenotypical change characterised by the up-regulation of CD31 and CD144 mRNA as well as a marked increase in sEVs secretion. MSC- and SH-MSC-derived sEVs had the capacity to internalise into endothelial cells, myoblasts and macrophages; exhibited a strong proangiogenic effect in vitro, particularly in promoting endothelial cell proliferation and pseudotube formation, likely due to an enriched cargo of angiogenic factors. These results highlight the dual benefit of hypoxia conditioning and endothelial differentiation of MSCs to optimise the angiogenic potential of their secreted sEVs, thus paving the way for innovative regenerative therapies in ischemic diseases.

Extracellular vesicles (EVs) have gained significant attention as emerging tools in diagnostics and therapeutics. Using the Gartner Hype Cycle framework, this commentary examines the current trajectory of EV research, from initial enthusiasm to growing concerns about reproducibility, standardization and clinical translation. We highlight key challenges, including EV heterogeneity, methodological inconsistencies and publication bias, which risk stalling progress. Ongoing efforts by the International Society for Extracellular Vesicles (ISEV), including Minimal information for studies of extracellular vesicles (MISEV) guidelines and the extracellular vesicle-transparent reporting and centralizing knowledge (EV-TRACK) database, have been crucial for advancing the field. We tackle actionable priorities to support rigorous, transparent and clinically meaningful EV research that would prompt the actual translation.