Journal of extracellular biology最新文献

Proteomic and transcriptomic analyses of cerebrospinal fluid (CSF)-derived extracellular vesicles (EVs) offer unique insights into molecular changes associated with central nervous system (CNS) diseases and may result in biomarker identification. No gold standard method to enrich EVs from CSF has been established, and head-to-head comparisons of outputs of different protocols are scarce. Using a large pool of CSF, we characterised the EV preparations resulting from four enrichment protocols and compared them in terms of yield and purity. We found that particles enriched by ultracentrifugation (UC) or a combination of ultrafiltration and size exclusion chromatography (UF-SEC) exhibited the typical morphological and biochemical characteristics of small EVs and were highly enriched in proteins and polyadenylated (polyA) transcripts associated with EV-related biological processes. UF-SEC preparations had higher particle yields, whilst more proteins were identified in UC preparations. Approximately 40% of the EV preparations’ proteome was not identified in unenriched CSF, among which a core proteome of 45 proteins was identified in 30 EV preparations from independent experiments, which may serve as CSF-derived EV markers. Enrichment scores to protein contaminants, albumin and apolipoprotein E were higher in UF-SEC preparations. In conclusion, all protocols analysed here resulted in enrichment of particles with small EV characteristics, with EV enrichments from UF-SEC resulting in the highest yield and purity.

Alzheimer's disease (AD) is a major neurodegenerative disorder that affects more than 55 million people, with an incidence that is projected to triple by 2050. Despite continuous advancements in the field, reliable treatment and early detection strategies remain elusive. Extracellular vesicles (EVs) play a major role in cellular communication throughout the body. In this study, we assessed the cargo of neuronal-specific EVs for their potential as AD biomarkers. We isolated EVs released from iPSC-derived excitatory glutamatergic neurons generated from eight AD patients (ADiNEVs) and six healthy controls (iNEVs). We performed RNA-sequencing and identified significant differences in RNA cargo between ADiNEVs and iNEVs. Notably, fewer small nuclear RNAs (snRNAs) were found in ADiNEVs. RNA transcripts significantly more abundant in ADiNEVs included MT-CO1, PRR32 and IGSF8 messenger RNAs. We also observed fewer XIST long noncoding RNAs and miR-7-5p microRNA content in ADiNEVs. These findings suggest that precision medicine approaches, such as characterising the content of EVs from a patient's own cells, could advance early detection and management of AD.

Extracellular vesicles (EVs) offer a minimally invasive approach for cancer detection and monitoring. However, the lack of standardized methods for clinical biospecimen preparation and EV isolation limits the clinical utility of EV-based biomarker assessments. A targeted need exists for detailed analysis of plasma EV content. Our study investigates the impact of clinical sample preparation and our ExoTIC device on the quality of plasma-derived EVs and their RNA/protein cargo in metastatic castration-resistant prostate cancer (mCRPC) patients. We assessed sample preparation variables: blood anti-coagulant choice (EDTA or sodium citrate), type of plasma platelet fraction (platelet-rich or platelet-poor), and use of protease inhibitors. EVs were isolated via ExoTIC device, followed by EV characterization and biomarker analysis using nanoparticle tracking analysis (NTA), cryogenic electron microscopy, Western blot, and digital PCR (dPCR). We detected mCRPC-relevant proteins (ARv7 and PSMA) in EVs from all plasma sample types with different sample preparation variables. Additionally, our findings indicate that platelet-poor plasma (PPP) is optimal for detecting EV- and biologically associated mCRPC biomarker miR-375. In this pilot study (n = 3), elevated EV miR-375 levels in PPP samples from mCRPC patients experiencing disease progression during docetaxel treatment were associated with poor therapeutic response to docetaxel chemotherapy, which aligns with our preceding in vitro and in vivo study. Optimal biospecimen preparation for EV analysis could enhance detection accuracy and patient management, highlighting detection of plasma EV-associated mCRPC-specific marker proteins (ARv7 and PSMA) and microRNA miR-375.

Dysfunction of vascular endothelium is characteristic of many aging-related diseases, including Alzheimer's disease (AD) and AD-related dementias (ADRD). Although it is widely posited that endothelial cell dysfunction contributes to the pathogenesis and/or progression of AD/ADRD, it is not clear how. A plausible hypothesis is that intercellular trafficking of extracellular vesicles (EVs) from senescent vascular endothelial cells promotes vascular endothelial cell dysfunction. To test this hypothesis, we compared the expression of proteins and miRNAs in EVs isolated from four sets of genetically identical early passage non-senescent (EP) versus late passage senescent (SEN) primary human coronary artery endothelial cells (HCAECs) derived from four donors. Proteomics and miRNA libraries constructed from these EV isolates were evaluated using FunRich gene ontology analysis to compare functional enrichment between EP and SEN endothelial cell EVs (ECEVs). Replicative senescence was associated with altered EV abundance and contents independent of changes in EV size. Unique sets of miRNAs and proteins were differentially expressed in SEN-ECEVs, including molecules related to cell adhesion, barrier integrity, receptor signalling, endothelial-mesenchymal transition and cell senescence. miR-181a-5p was the most upregulated miRNA in SEN-ECEVs, increasing >5-fold. SEN-ECEV proteomes supported involvement in several pro-inflammatory pathways consistent with senescence and the senescence-associated secretory phenotype (SASP). These data indicate that SEN-ECEVs are enriched in bioactive molecules implicated in senescence-associated vascular dysfunction, blood–brain barrier impairment, and AD/ADRD pathology. These observations suggest involvement of SEN-ECEVs in the pathogenesis of vascular dysfunction associated with AD/ADRD.

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.