Journal of extracellular biology最新文献

Chronic kidney disease (CKD) and obesity are major chronic diseases in the United States. Although obesity is a risk factor for CKD, little is known about how obesity contributes to CKD. Due to their role as intercellular communicators, extracellular vesicles (EVs) may be a factor connecting obesity and CKD. Circulating cell-free mitochondrial DNA (ccf-mtDNA), a damage-associated molecular pattern molecule associated with inflammation, is associated with renal disease and may be encapsulated within EVs. In this longitudinal study, we isolated plasma EVs and analysed EV mtDNA levels in a cohort of African American and White obese middle-aged individuals who at visit 1 did not have CKD but developed CKD by visit 2 (n = 19; CKD group) and matched this group to controls who did not develop CKD by visit 2 (n = 56; control group). In our cross-sectional analyses at visit 1, we found significant interactions for EV mtDNA levels between race and CKD status, poverty status and CKD status, and sex and CKD status. EV mtDNA levels were significantly lower in participants within the African haplogroup who developed CKD compared to participants within the European haplogroup who developed CKD and the African haplogroup control group. In our longitudinal analyses using data from both visit 1 and visit 2, individuals who developed CKD had lower EV mtDNA levels. Stratification by haplogroup showed that among participants within the African haplogroup, those who developed CKD had significantly lower EV mtDNA levels than those in the control group. In conclusion, EV mtDNA levels were lower in individuals who develop CKD. Our findings demonstrate that CKD status and mtDNA haplogroup influence EV cargo in obese individuals.

Extracellular vesicles (EVs) contain a variety of biomolecules, including DNA, RNA, lipids and proteins. They can interact with target cells to perform various functions, offering potential for therapeutic applications like drug delivery and diagnosis. The growing interest in EVs drives the need for robust methods for EV characterisation. One of the prevalent EV characterisation methods is scatter-based nanoparticle tracking analysis (Sc-NTA). This method measures the size and concentration of particles by tracking the scattered light from individual particles. However, Sc-NTA has limitations in selectivity, as it detects all scattered light and fails to distinguish EVs from other nanoparticles, such as protein aggregates. To overcome this limitation, fluorescence-based NTA (Fl-NTA) is being utilised, where fluorescence tagging is used to selectively detect EVs. In previous studies, lipophilic dyes were employed for membrane labelling, but this resulted in false-positive signals due to the staining of even non-vesicular extracellular particles (NVEPs). Immunolabelling methods using antibodies that specifically bind to EV-specific protein were also introduced; yet challenges with sensitivity and photostability of the organic dyes remained. To address the challenges, we conjugated quantum dots (QDs) to antibodies that specifically bind to EV-specific markers, CD9, CD63 and then immunolabelled the EVs. Labelling conditions were optimised to develop a robust protocol for QD-based immunolabelling. Detection sensitivity was evaluated by comparing QD-based immunolabelling with Alexa dye-based methods. Furthermore, size distribution analysis demonstrated the ability of QDs to detect smaller EV populations. Finally, subpopulations of EVs from various cell lines were profiled. This approach enhances the accurate characterisation of EVs, providing a reliable and reproducible method for EV quality control and improved insights into their heterogeneity.

Human mesenchymal stem cells (hMSCs) have been under investigation in preclinical and clinical settings for treating neurological disorders in recent years. Predominantly due to paracrine effects in vivo, hMSC-secreted extracellular vesicles (EVs) are at the forefront of these investigations. In this study, the therapeutic efficacy of hypoxia hMSCs and the secreted EVs labelled with iron oxides was evaluated in a preclinical model of ischemic stroke. Transcriptome and proteomics analysis of hMSCs under hypoxia indicated alterations in metabolic pathways and EV biogenesis. Hypoxia preconditioning increased EV yield by 57% with similar EV size and exosomal marker expression. EV cargo analysis using proteomics and microRNA-sequencing revealed that hypoxia preconditioning upregulated expression of metabolic proteins related to hypoxia-inducible factor signalling, neurogenesis and EV biogenesis. Magnetic resonance imaging following in vivo administration of iron oxide-labelled hMSCs and EVs provided assessment of biodistribution and therapeutic efficacy. The results indicated differential recovery in sodium levels in rats following hMSC and EV administration compared to the vehicle-only group, supported by lactate levels and functional assessment. hMSC-EVs localized to the ischemic lesion and evoked a therapeutic response after a single bolus injection. This study has significance in developing human stem cell-free therapeutics for treating ischemic stroke.

Urinary extracellular vesicles (uEVs) are well-known to express tetraspanin family membrane proteins abundantly on their surface. In this study, we aimed to develop an upconverting nanoparticle (UCNP)–based lateral flow immunoassay (UCNP-LFIA) designed for the rapid and high-sensitivity detection of CD63-positive uEVs for direct urinalysis. The assay utilizes UCNPs reporter to detect low concentrations of EVs. Minimally processed uEV samples from bladder cancer (BlCa) (n = 62), benign prostatic hyperplasia (BPH) (n = 50) and healthy (n = 30) individuals were tested in sandwich UCNP-LFIA format, capturing uEVs with the same anti-CD63 antibody conjugated to UCNP and immobilized on the test zone. After 80 min, the strips were read with an upconversion luminescence reader device. This UCNP-LFIA measured CD63-positive EVs with high sensitivity, exhibiting a limit of detection (LoD) of 4 × 10⁷ EVs/mL. The concentration of CD63-positive EVs in BlCa patients showed a 2.3-fold increase compared to benign conditions (p = 0.007), and a 16-fold increase compared to healthy controls (p = 0.00001). The results demonstrate the potential of UCNP-LFIA platform for sensitive and quantitative detection of uEVs, highlighting its promise as a tool for EV detection at point-of-care diagnostics.

Extracellular vesicles (EVs) are increasingly recognized as promising disease biomarkers and therapeutic carriers. However, standardizing blood-derived EV isolation remains challenging due to the heterogeneity of EV populations and variability among isolation techniques. In this study, we systematically evaluated three distinct EV isolation methods, including asymmetrical flow field-flow fractionation (AF4), size-exclusion chromatography (SEC) and automated centrifugal microfluidic disc system combined with functionalized membranes (Exo-CMDS), to compare their efficiency in isolating EVs from both freshly frozen and freeze-thawed plasma samples. We utilized an integrative approach combining Proximity-dependent Barcoding Assay (PBA) for single-EV surface protein profiling, Liquid Chromatography–Mass Spectrometry (LC-MS/MS) for bulk proteomic analysis, along with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) to assess EV yield, morphology, surface protein expression and subpopulation diversity. Our results revealed significant differences in three EV isolation methods. AF4 is particularly enriched for EV subpopulations expressing high levels of classical tetraspanins (e.g., CD81, CD9 and CD151), and single-pass membrane proteins (e.g., ITGA4 and ITAGB1). Exo-CMDS demonstrated the highest reproducibility across samples, isolating specific EV subpopulations enriched in markers like CD5. SEC provided the highest yield but co-isolated significant amounts of non-vesicular particles, including lipoproteins. The findings contribute valuable insights toward standardized and reliable EV isolation practices for research and clinical applications.

Atopic dermatitis (AD) is a chronic inflammatory disease characterized by severe itching and eczematous lesions. Despite various treatments, AD patients experience side effects and fail to achieve full remission. This study investigated the therapeutic potential of extracellular vesicles (EVs) derived from IFN-γ-primed induced mesenchymal stem cells (IFN-γ-iMSC-EVs) in a 2,4-dinitrochlorobenzene (DNCB)-induced AD mouse model. We also examined whether IFN-γ-iMSC-EVs could suppress IL-4/13-induced Th2 responses in keratinocytes. The therapeutic outcome of IFN-γ-iMSC-EVs was comparable to or more effective than baricitinib or clobetasol. While severe weight loss was observed in mice treated with clobetasol, no significant weight reduction occurred in those receiving IFN-γ-iMSC-EVs. Histological analysis demonstrated reduced skin thickness, decreased infiltration of mast cells and inflammatory cells, and suppression of the Th2 immune response, as evidenced by decreased signalling of IL-4, IL-13, and IL-31. IFN-γ-iMSC-EVs also led to a greater reduction in inflammation and pruritus compared to baricitinib and clobetasol. Additionally, skin barrier integrity and epidermal protein expression were improved in IFN-γ-iMSC-EVs. In IL-4/13-stimulated keratinocytes, the decrease in JAK1/2 gene expression and the increase in Keratin 1 gene expression were more prominent in IFN-γ-iMSC-EVs than in baricitinib. The results suggest that IFN-γ-iMSC-EVs have the potential to inhibit AD progression and represent a novel therapeutic option for AD.

Extracellular vesicle (EV) secretion and cargo composition are dysregulated in metabolic diseases. This study aimed to investigate how changes in serum EV concentration and protein composition reflect the metabolic effects of a high-fat diet (HFD) and time-restricted feeding (TRF), with a particular focus on adipocyte-derived EVs (Ad-EVs) in circulation. Mice were fed an HFD for 18 weeks prior to being placed either ad libitum or on a TRF for an additional 10 weeks. Mice on a normal chow ad libitum served as the control. The TRF group had food available for 10 h and fasted for 14 h per day. The serum EV size profile and amount displayed sex- and age-dependent changes in HFD-induced obesity, with age reducing EV amounts. HFD decreased small EV populations and increased larger EV populations, while TRF reversed these changes. Quantitative proteomic analysis showed that the abundance and composition of EV proteins changed in response to both acute stimulation with lipopolysaccharides (LPS) and HFD. Gene ontology analysis identified specific sets of EV proteins and their involved biological processes, reflecting the effect of LPS and HFD, as well as the reversal effect of TRF on metabolic and inflammatory pathways. EV proteins altered by HFD and those reversed by TRF had low protein overlap but significant functional overlap in biological processes. TRF activated the PPAR signalling pathway and the AKT-mTOR signalling pathway. The most significant impacts of HFD and TRF were observed on lipoprotein and carbohydrate metabolism, the complement system, and neutrophil degranulation. Additionally, we showed that serum Ad-EVs respond dynamically to HFD and TRF. Our findings suggest that EVs play a role in diet-induced metabolic and inflammatory responses, with changes in circulating EVs, particularly Ad-EVs, reflecting metabolic adaptations to dietary exposures and interventions.

Small extracellular vesicles (sEVs) possess many advantageous characteristics which highlight their potential as nanocarriers for biomedical applications, including the ability to cross the blood brain barrier, improved biocompatibility and exhibit tissue tropism. Despite this potential, the clinical translation of sEVs has been hindered by a variety of factors and lipid nanoparticles (LNPs) remain as the gold standard for nanocarriers, indicating a knowledge gap which could unlock the potential of sEVs. A growing body of research suggests that the lipid profile, rather than the proteome, of sEVs may be contributing to these beneficial characteristics much more than previously thought. This review highlights and discusses the current state of the field in terms of lipid composition between sEVs originating from various cell sources and the roles which the different lipids play in the function of sEVs as natural nanocarriers within the body. We also discuss the potential of various EV-mimetics and synthetic EVs (synEVs) in terms of clinical translation which may provide a means to allow wider therapeutic adoption of EVs.

Immune checkpoint inhibitors (ICI) have revolutionized the treatment of metastatic malignancy. However, unique immune response patterns can occur, such as pseudoprogression, which corresponds to new lesion development or temporary tumour growth followed by regression. Misidentifying pseudoprogression may halt ICI therapy, due to the absence of biomarkers to distinguish progression from pseudoprogression. In 2020, our team proposed small extracellular vesicles expressing PD-L1 (sEV-PD-L1) as a predictor of melanoma treatment response. We report a case of pseudoprogression in a patient treated with nivolumab and ipilimumab for metastatic melanoma, and showing reduced circulating sEV-PD-L1. To our knowledge, this is the first report of PD-L1 monitoring in circulating sEV during pseudoprogression under ICI. A decrease in PD-L1 in circulating sEV might be an early sign of disease response to ICI, and may help to diagnose pseudoprogression. This case supports further evaluation of sEV-PD-L1 to identify responder patients to ICI, especially in case of pseudoprogression.

Trial Registration: EXOMEL1 P/2018/40 1 AC-2015-2496/DC-2014-2086. NCT05744076.

Extracellular vesicles (EVs) are nanosized, membrane-bound particles released by virtually all cell types, serving as messengers within tissues and across organs via the bloodstream. EVs encapsulate diverse molecular cargo that reflects the phenotypic state of their originating cells, making them promising candidates for liquid biopsy applications. However, the heterogeneity of circulating EVs, comprising particles from various cell types and non-vesicular entities like lipoproteins, poses significant challenges for isolating tissue-specific EV populations. This review examines current methodologies for detecting and isolating tissue-specific EVs from blood, focusing on immunoaffinity capture (IAC) strategies that leverage surface marker expression for specificity. Key considerations, including the selection and validation of markers, are discussed alongside advances in EV subtyping and isolation protocols. Challenges such as marker cross-reactivity, EV biogenesis and transport dynamics are highlighted to underscore the complexity of achieving clinical utility. By providing an overview of validated tissue-specific markers and isolation techniques, this review aims to facilitate the development of EV-based biomarkers with enhanced specificity and sensitivity, enabling minimally invasive monitoring of organ function and disease.