Journal of Extracellular Vesicles

Extracellular vesicles (EVs) are small lipid structures secreted by cells that originate from the cell surface (typically enriched in the tetraspanin (tspan) CD9) or from multivesicular bodies (typically enriched in the tspan CD63). Current methods for studying EVs involve concentrating and purifying EVs, without providing information about the distance or amount of EVs that may transfer from one cell to another. Here, we developed a coculture assay of human mammary MCF-7 cells to study the transfer of mCherry-CD81 or mCherry-CD9 from "donor" cells to a lawn of "acceptor" cells stained with cell tracker blue or green (CTB/CTG), non-transferrable fluorescent dyes. Using confocal fluorescence microscopy, we observed the presence of spots containing mCherry-CD81 or mCherry-CD9 outside donor cells, concentrated at short distance from donor cells and that overlapped with CTB signal, suggestive of their internalization in acceptor cells. Endogenous CD63, CD81 and CD9 also transferred more efficiently at short distances, even in the presence of a flow, as shown by immunostaining cocultures of wild type and KO CD-63, or -9, or -81 cells with antibodies directed against these tspans. Computation of the (x,y,z) coordinates of tspans-containing spots revealed a double polarized transfer: in (x,y), it distributed along a gradient that started from donor cells and decreased with the distance, and in (z), it was stronger in basal compared to upper planes, a (z) polarization that was affected by syntenin-1 depletion in donor cells. Simultaneous monitoring of CD9/CD81 transfer from into double CD81/CD9 KO cells showed that cells transferred more CD81 spots than of CD9. At the basal level, CD63 and CD81 spots were plasma membrane derived as they almost always contained CD9+, and resembled membranous remnants of migration. However, live cell imaging showed migration independent secretion of EVs in the extracellular space, in upper planes. Altogether, not only is our coculture assay suitable for the direct qualitative and quantitative study of EV-transfer, but it highlighted shared three-dimensional features of EV markers transfer between cells.

Kidney transplantation faces a critical paradox: while thousands await organs, approximately 30% of potential deceased donor kidneys are discarded for various reasons, including subjective assessments due to the lack of an objective molecular biomarker of preservation quality. Here, we applied novel "top-down" proteoform imaging mass spectrometry across living donor (LD), deceased donor (brain death or cardiac death), and discarded human kidneys to quantify proteoforms correlating with post-transplant kidney function. This approach preserves post-translational modifications and splice variants, revealing molecular tissue variability beyond protein presence. LD kidneys displayed robust metabolic signatures, including L-xylulose reductase and cytochrome oxidase subunits, whereas deceased donor and discarded organs showed elevated cellular stress markers such as alpha-B-crystallin and peroxiredoxin 1. Post-transplant blood proteoform analysis validated tissue findings, demonstrating persistent cellular stress and immune activation in deceased donor recipients compared with physiologic wound healing in LD recipients. Consistent with these molecular predictions, serum creatinine levels were highest in DCD, intermediate in DBD, and lowest in LD recipients. The intersection of tissue proteoform signatures across all marginal tissues identified four proteoforms consistently elevated in deceased and discarded kidneys: ACTG1, acetylated CRYAB, PARK7, and S100A4. Collectively, these proteoforms capture key molecular indicators of graft quality, reflecting oxidative stress, cellular injury, and immune activation pathways. As such, they represent promising point-of-care (POC) biomarker candidates for objective kidney classification, potentially improving donor kidney utilization. Translational statementCurrent methods for evaluating donor kidney quality rely on subjective assessments, contributing to the discard of approximately 30% of potentially viable organs. This study demonstrates that "top-down" proteomics can objectively identify molecular signatures distinguishing high-quality from marginal donor kidneys. Top-down proteomics analyzes intact proteins with their post-translational modifications or cleavage products, termed proteoforms to provide mechanistic insights into graft quality. We identified four proteoforms (ACTG1, acetylated CRYAB, PARK7, and S100A4) to be consistently elevated in deceased and discarded kidneys, reflecting oxidative stress, cellular injury, and immune activation. These molecular markers correlated with post-transplant kidney outcome, as measured by serum creatinine levels and recipient blood proteoforms. As a next step, validation in larger cohorts could establish these proteoforms as point-of-care biomarkers for real-time donor kidney assessment during procurement. This objective molecular stratification could reduce unnecessary organ discards and improve transplant outcomes by matching organ quality with recipient risk profiles.

Extracellular vesicles are key intercellular messengers that modulate the function of target cells by carrying effectors, either at their surface or in their lumen. In the latter case, their action depends on the ability to deliver their content into the cytosol of target cells. How efficiently EVs deliver their content upon interaction with their target cell is thus a central question for understanding the functional impact of this mode of action. To address this question, signal-driven bimolecular interactions between two partners located respectively in the EV lumen and the target cell cytosol have become a widely used strategy to detect the cytosolic delivery EV content. However, the detection of cytosolic delivery with these assays was often tributary to the artificial enhancement of the fusion between EV and cell membranes, through for instance VSV-G fusogenic protein expression. Here we provide a robust and quantitative LUCiferase-based complementation assay (HiBiT/LgBiT), to quantify the Internalization and cytosolic Delivery of EV content: LUCID-EV. By optimizing the signal-to-noise ratio of the assay, the method for loading HiBiT fragment into EVs (fusion to a lipid-binding domain rather than to tetraspanins), and the intracellular position of LgBiT (associated to membranes), we could quantify cytosolic delivery from various non-VSV-G-expressing EVs into target immune dendritic cells. Importantly, this delivery did not involve the acidic late endosomes environment required for VSV-G-dependent EV cytosolic delivery. The limited efficacy of the process highlights the need for highly sensitive assays like the one described here. Further development of the LUCID-EV assay could help identifying EV/target cells pairs with enhanced cytosolic delivery properties and characterize the cellular route for delivery.

The intestinal microbiome influences immune recovery and long-term outcomes after allogeneic hematopoietic stem cell transplantation (allo-SCT). While reduced bacterial diversity and depletion of immunomodulatory microbial metabolites during peri-engraftment have been linked to acute graft-versus-host disease (aGvHD) and mortality, it remains unclear whether microbiome recovery after engraftment and immune reconstitution is better reflected by bacterial diversity or by microbial metabolic output. We aimed to define microbiome recovery in the late post-transplant period and test whether a metabolite-based biomarker improves the prediction of clinical outcomes, including overall survival (OS) and chronic (c) GvHD. In this two-center longitudinal observational study, serial stool samples were collected from pre-transplant baseline to day +100 after allo-SCT in a discovery cohort (n = 20, Technical University Munich University Hospital (TUM)) and an independent validation cohort (n = 100, University Hospital Regensburg (UKR)). Gut microbiome composition was assessed by 16S rRNA gene amplicon sequencing, with metagenomic profiling in selected patients, and stool metabolites were quantified using targeted mass spectrometry. Patients were classified as RECOVERY or NO RECOVERY based on changes in bacterial richness between baseline and the post-transplant period. To capture microbial metabolic output, the previously established Immune-Modulatory Metabolite Risk Index (IMM-RI), comprising butyric, propionic, and isovaleric acids, desaminotyrosine and indole-3-carboxaldehyde, was adapted to the late post-transplant period (IMM-RI post-TX). Bacterial alpha diversity frequently improved by day +100; however, this did not consistently indicate restoration of baseline community structure and was not paralleled by recovery of stool metabolite profiles. Accordingly, RECOVERY status showed a limited association with survival or transplant-related mortality (TRM). In contrast, IMM-RI post-TX low-risk identified patients with preserved butyrate-associated biosynthetic capacity and was significantly associated with improved OS in both cohorts (UKR: HR 0.2052, 95% CI 0.07703 - 0.5466, p < 0.0001). In the validation cohort, IMM-RI post-TX low-risk was significantly associated with reduced relapse-related mortality. Interestingly, stool butyric-, propionic and valeric acid concentrations were increased in cGvHD of the skin, indicating context-dependent metabolite effects. These findings suggest that metabolite profiling outperforms bacterial diversity for predicting outcomes after allo-SCT and support microbial metabolites as promising biomarkers for risk stratification and actionable candidates for precision microbiome interventions after allo-SCT.

Efficient, aggregation-free extracellular vesicles (EVs) labeling is essential for studying their dynamics in-vitro and in-vivo. However, traditional dyes introduce limitations including aggregation, membrane intercalation, fluorescence transfer and inconsistent performance across EV sources thus distorting quantification, altering surface properties and confounding uptake and biodistribution analyses. Here, we systematically evaluated CytoLight, a luminal dye traditionally used for live-cell imaging, as an alternative for EV quantification, characterization, uptake analysis and in-vivo tracking, benchmarking it against PKH26, CFSE and ExoBrite across multiple platforms. CytoLight generated stable, intravesicular fluorescence without aggregation or membrane alteration, eliminating artifacts characteristic of conventional dyes. Using fluorescence-NTA and single-EV flow cytometry, CytoLight showed more consistent labeling across EV types than CFSE or ExoBrite, while avoiding PKH-related micelle-driven artifacts and exhibited compatibility with CD81 dual-detection. In uptake assays, CytoLight produced EV-specific endocytosis-dependent internalization signals exceeding labeled-BPS/protein controls. In-vivo, CytoLight-labeled EVs enabled fluorescent biodistribution mapping showing conventional EV tropism patterns distinguishable from labeled-PBS controls. These findings establish CytoLight as an effective, aggregation-free EV-labeling strategy. Its stability, specificity, compatibility with single-EV platforms and reliable performance in both cellular uptake and biodistribution studies position CytoLight as a practical, scalable alternative to current dyes, providing a stronger foundation for standardized and reproducible EV research.

Extracellular vesicles (EVs) are critical mediators of intercellular communication, carrying molecular cargos such as small noncoding RNAs (ncRNAs) that reflect the physiological and pathological state of their cells of origin. However, studying brain-derived EVs has been challenging due to the blood-brain barrier. Here, we optimized and validated an open-flow microdialysis (OFM) protocol for sampling EVs directly from brain interstitial fluid (ISF) in wild-type and APP/PS1 transgenic mice. Ex-vivo validation using plasma EVs demonstrated that OFM effectively captures the full EV population. In-vivo cerebral OFM (cOFM) enabled successful collection of brain ISF EVs, which were characterized by nanoparticle tracking analysis (NTA), electron microscopy, and western blotting, confirming their similarity to EVs isolated directly from brain tissue and plasma. Identification of small ncRNA cargos revealed that EVs sampled from brain ISF by cOFM were enriched in brain-specific signatures, many of which are associated with neuronal cell populations and biological functions. Furthermore, we observed a unique small ncRNA signature from the brain ISF EVs in the Alzheimers disease preclinical model compared to wild-type mice. These small ncRNAs were associated with genes considered important in biological functions associated with neurodegeneration. Our findings demonstrate that cOFM is a powerful tool for in-vivo sampling of brain EVs and highlight the unique molecular landscape of ISF EV small ncRNA cargos. This study offers new opportunities for biomarker discovery and mechanistic insights into neurodegenerative diseases, such as Alzheimers disease.

Extracellular vesicles (EVs) are key mediators of intercellular communication, yet their molecular profiles across tissues and species remain poorly characterized, particularly due to currently available methods requiring a large amount of biological material (tissue or biofluids). Here, we established a workflow allowing the deep phenotyping of EV cargos starting from single samples of human and mouse origin. We took advantage of standardised EV isolation procedures and multi-omic techniques for the isolation and analysis of EVs from brain and plasma of human and mouse, integrating flow cytometric profiling, proteomics, miRNA sequencing, and fatty acid profiling. Here we report specific brain-derived EVs proteome, enriched in neuronal and glial proteins, polyunsaturated fatty acids profiles, and distinct miRNAs. At the periphery, we also report plasma-derived EVs signatures reflecting immune, metabolic, and systemic transport functions. Despite these expected material-specific differences, EVs from the same source displayed greater similarity across species than EVs from different material, supporting the translational relevance of mouse models. Importantly, using state-of-the-art miRNA profiling approach, we identified novel EV-specific miRNAs in human and mouse brain EVs, potentially allowing the exploration of new roles in neuronal signalling. Overall, we report here a method enabling deep multi-omic characterization from minimal starting material, offering a practical approach for studies with limited biological samples. These findings also demonstrate that the origin strongly shapes EV composition, highlighting conserved and species-specific molecular features, and provide a scalable framework for multi-omic investigations of EV biology. Summary StatementWe present a standardised workflow allowing multi-omic profiling of brain and plasma-derived EVs from minimal human and mouse material. Our findings reveal both tissue-specific and species specific EV molecular signatures.

BackgroundExtracellular vesicles (EV), secreted membrane particles involved in cell-to-cell communication, carry important information on immunity and its dysregulation. Recent studies have demonstrated the crucial role of peripheral and central inflammation in causing Parkinsons disease (PD), as well as the involvement of EV in mediating neuron-glial interactions during neurodegeneration. However, the underlying mechanism of plasmatic EV in PD pathogenesis remains unknown. MethodsEV were isolated from pool of plasma of PD patients and age- and sex-matched healthy controls (HC) using size-exclusion chromatography and characterized by nanoparticle tracking analysis, western blot, and transmission electron microscopy. SH-SY5Y neurons and HMC3 microglia cells were treated with EV, and their impact was evaluated using flow cytometry and immunofluorescence. Conditioned medium (CM) from EV-treated HMC3 cells was applied to SH-SY5Y neurons to determine indirect neurotoxic effects. Cytokine profiling and senescence-like features of EV-treated HMC3 cells were assessed. Unbiased proteomic analysis of PD-EV and HC-EV were further performed. ResultsPD-EV induced axonal degeneration and cell death in SH-SY5Y neurons and increased levels of TNF-, IL-1{beta}, IFN-{gamma}, IL-8, and CCL11, accompanied by the expression of p16INK4a in HMC3 cells, suggesting a proinflammatory, senescence-associated secretory phenotype (SASP). Enrichment pathway analysis revealed that these changes were mainly related to inflammatory and immune responses. Moreover, CM from PD-EV-HMC3 cells increased apoptotic cell death in SH-SY5Y neurons more than direct PD-EV. Notably, proteomic analysis of PD-EV showed higher expression of proteins involved in complement cascades, immune response, phagocytosis, and post translational protein translation, further supporting the potential of EV to induce inflammatory changes in PD. ConclusionsThis study demonstrates that plasmatic PD-EV contributes to neuronal degeneration by reducing neuronal integrity and indirectly by activating microglia through the secretion of pro-inflammatory, senescence-associated mediators. Circulating EV exerts a role in bridging peripheral inflammation with microglia, modulating neuroinflammatory events.

BackgroundIn lung transplantation, de novo immunodominant donor-specific anti-HLA antibodies recognizing HLA-DQ antigens (dn-iDSA-DQ) are predominant and can induce chronic lung allograft dysfunction (CLAD). We previously developed a method to measure the active concentration of dn-iDSA-DQ. We aimed to determine whether this new quantitative biomarker is associated with transplantation outcomes. MethodsThis retrospective multicentre cohort study included 90 lung transplant recipients (LTRs) developing dn-iDSA-DQ, evidenced through single antigen flow beads (SAFB) follow-up. We measured the active concentration of dn-iDSA-DQ at the time of their first detection (T0) for all LTRs, and within the 2 years after DSA detection, whenever possible. SAFB dn-iDSA-DQ characteristics and clinical data were retrieved up to 5 years after DSA detection. ResultsWe tested 184 sera with SPR (n=90 at T0, n=94 within the 2 years after DSA detection), among which 63 (34.4%) had a quantifiable concentration of the dn-iDSA-DQ ([&ge;]0.3 nM). The median SAFB mean fluorescence intensity (MFI) of the dn-iDSA-DQ with a concentration [&ge;]0.3 nM was higher (p<0.0001), yet the correlation between SAFB MFI and active concentration was low (r=0.758, p<0.0001). In multivariate analysis, a concentration of the dn-iDSA-DQ [&ge;]0.3 nM at T0 was independently associated with a lower 2-year CLAD-free survival (HR 2.06, p=0.02). A concentration of the dn-iDSA-DQ [&ge;]0.3 nM within the 2 years from DSA detection was associated with a lower graft survival in univariate analysis. ConclusionsActive concentration of dn-iDSA-DQ appears as a valuable biomarker to identify pathogenic DSA at their first detection because of its association with CLAD.

Accurate characterization of the nanoparticle (NP) protein corona is essential for predicting biological fate, safety, and therapeutic efficacy, and for enabling robust biomarker discovery. Standard isolation techniques, most commonly centrifugation and magnetic separation, are widely used, yet they rarely account for co-isolating endogenous biological NPs such as extracellular vesicles (EVs). This oversight can distort the apparent "biological identity" of the NP. Here, we quantitatively demonstrate the magnitude and impact of EVs on the perceived protein corona composition. We incubated highly monodisperse polystyrene NPs (50-1000 nm) and superparamagnetic beads in either standard human plasma or plasma depleted of EVs by immunoaffinity capture targeting 37 EV surface epitopes. Mass spectrometry revealed that EV depletion reduced the number of proteins identified on polystyrene NPs by 60-75% and on magnetic beads by 45-50%. Importantly, EV depletion also altered the apparent abundance hierarchy; it restored the expected relative abundance and rank of major plasma proteins such as albumin and shifted the top-ranked proteins from intracellular cytoskeletal component, consistent with EV carryover, to genuine soluble plasma adsorbates (e.g., apolipoproteins, complement factors). These results highlight that standard corona workflows can inadvertently co-isolate a vast array of EV-associated proteins, yielding inaccurate proteomic profiles. Discriminating genuine corona proteins and EV-associated contaminants is critical for advancing nanomedicine, ensuring predictive safety and efficacy profiles, and enhancing the precision of NP-based biomarker discovery.

Extracellular vesicles (EVs) mediate cell-to-cell communication and are considered potential drug delivery vehicles. Nevertheless, whether EV-embedded cargo can be efficiently delivered into the cytosol of recipient cells remains debated. Here, we investigated the fate of syntenin, a well-established internal cargo of small EVs (sEVs). Using quantitative assays, we show that [~]85% of internalized sEV-embedded syntenin can be delivered to the cytosol of recipient cells within short periods of time. Yet, even at low dose, we find that the internalization of sEVs carrying syntenin is rather inefficient ([~]0.03% of the administered dose). Moreover, we observe that the capture of sEVs by recipient cells is non-saturable over time and largely more efficient than their internalization. Finally, we identify the N-terminal domain of syntenin and the phosphorylation state of a Src-targeted tyrosine residue in this domain, as key determinants for its incorporation into sEVs that support cytosolic delivery. These findings challenge, current views in the field by indicating that sEV internalization may be a marginal process (on the contrary to capture) and that cytosolic delivery can be highly efficient. Moreover, our study identifies molecular determinants governing cytosolic delivery of sEV-embedded syntenin.

BackgroundThe efficacy of immune checkpoint blockade relies on the robust priming of T cells by immunostimulatory dendritic cells (DCs). However, the tumor microenvironment (TME) frequently drives DCs into a dysfunctional, pro-tolerogenic state governed by aberrant metabolic rewiring, creating a barrier to durable antitumor immunity. While tumor-derived extracellular vesicles (EVs) are abundant in the TME, their specific role in orchestrating this immunosuppressive metabolic reprogramming remains poorly understood. This study provides insight into the signaling axes through which tumor-derived EVs alter DC function and evaluates the therapeutic potential of targeting these pathways to overcome immunotherapy resistance. MethodsTumor models were engineered to express EV fluorescent markers to track tumor EV uptake in vivo. Bulk and single-cell RNA sequencing was integrated with multi-parameter flow cytometry to characterize the reprogramming of tumor EV-educated DCs both in vitro and in vivo. Western blotting, quantitative real-time polymerase chain reaction assays, various cellular metabolic assays, as well as T cell-based immunologic studies were utilized to characterize the underlying mechanisms of tumor EV-mediated DC reprogramming. DC-specific Ppara-deficient mice were developed to verify these mechanisms in vivo. PPAR- targeted inhibitors were evaluated based on their ability to overcome checkpoint inhibitor resistance in an autochthonous model of melanoma. ResultsTumor-derived EVs were found to promote tumor progression by suppressing host immunity. Further studies reveal that tumor-derived EVs induce a tolerogenic mregDC transcriptional signature characterized by the upregulation of immunoregulatory molecules in DCs both in vitro and in vivo. These tumor EV-educated DCs exhibit an impaired capacity for CD8+ T cell priming, while demonstrating a proficiency for promoting CD4+FoxP3+ regulatory T cell differentiation. Mechanistically, tumor EVs concurrently trigger the unfolded protein response (UPR) via the PERK-ATF4 and IRE1-XBP1s signaling axes, subsequently activating the SREBP2 and PPAR- transcription factors, respectively. This process drives both aberrant lipid accumulation and fatty acid oxidation (FAO) in DCs residing within the TME. DC-restricted ablation of PPAR- significantly reversed the pro-tolerogenic effect of tumor EVs in vivo while pharmacologic targeting of PPAR- overcomes anti-PD-1 resistance and augments CD8+ T cell infiltration in an autochthonous model of melanoma. ConclusionsTumor EVs contribute to the development and pro-tolerogenic function of mregDCs in the TME by triggering the UPR pathway. Aberrant lipid metabolism involving enhanced FAO are common characteristics associated with DC dysfunction in the TME. Strategies to interrupt these pathways represent promising approaches for reversing immune tolerance and enhancing tumor-targeted CD8+ T cell responses.

Extracellular vesicles (EVs) are lipid-bound particles that transfer cargos between cells. While plasma neuronal-derived EVs (NEVs) from individuals with mild cognitive impairment (MCI) and Alzheimers disease (AD) have been reported to exhibit high pathogenic potential, this study examined the impact of astrocyte-derived EVs (AEVs) in an aged AD mouse model. Plasma AEVs were isolated from cognitively normal control (CNC), MCI, and AD individuals using GLAST-based immunocapture and AEV size, purity, and tetraspanin were validated by flow cytometry, nanoparticle tracking, and super-resolution microscopy. AEVs pooled by clinical cohort were injected into the hippocampus of 6-month-old female PSAPP mice. Behavioral, biochemical, and neuropathological outcomes were assessed 6 months later. Rotarod assessment revealed significant impairment in motor coordination (p<0.0001) in mice receiving MCI- and AD-AEVs compared with those receiving CNC-AEVs. Morris water maze (MWM) also demonstrated cognitive deficits in MCI- and AD-AEV injected mice; however, no overt changes were observed in the staining of amyloid plaque burden (6E10), and neuroinflammation (GFAP). Immunoblotting of 82E1 and 22C11 confirmed A{beta}/AAP levels remained similar across all injected mice, whereas increased cortical tau accumulation was observed in MCI- and AD-AEV injected mice. Cerebellar synaptic density (SY-38) remained unchanged. While plasma AEVs from healthy individuals may confer neuroprotective benefits, AEVs from cognitively impaired individuals promoted tau accumulation in an amyloidogenic mouse model. These findings suggest that AEVs may play a dual role in AD as both potential biomarkers and mechanistic drivers of neurodegeneration, highlighting their relevance as targets for future therapeutic strategies.

Small extracellular vesicles (sEV) are membrane-enclosed nanoparticles found in body fluids that carry molecular cargo from their cells of origin. Their stability and disease-associated molecular signatures make them promising targets for the development of non-, or minimally invasive liquid biopsies, yet scalable approaches enabling single-vesicle quantification of sEV while resolving their heterogeneity remain limited. Here, we present PICO (Protein Interaction Coupling), a reference-free quantitative assay adapted for sensitive multiplex profiling of individual intact vesicles. PICO detects vesicle markers by requiring colocalization of two or more copies of the same protein or of distinct proteins (e.g., CD9 or CD9/CD63) on individual vesicles, using DNA-barcoded antibodies and digital PCR (dPCR) for quantitative readout. We demonstrate that this unique architecture of the assay provides high specificity by distinguishing EV-bound proteins from soluble counterparts, and can be adapted to target either surface-exposed or intravesicular biomarkers. PICO requires minimal sample input (1 {micro}l) and no specialized instrumentation beyond standard digital PCR. In a head-to-head comparison with nano-flow cytometry, PICO achieved a comparable limit of detection for sEV subpopulations. Profiling sEV isolates from blood for canonical markers (CD9, CD63 and CD81) and HER2 demonstrates precise, high-resolution quantification of sEV subpopulations in complex clinical samples and supports integration of scalable single-EV analysis into research and diagnostic workflows.

BackgroundP-glycoprotein (P-gp/ABCB1) is a key efflux transporter that maintains barrier integrity by clearing xenobiotics and toxic metabolites. At the feto-maternal interface, trophoblast-derived extracellular vesicles (CTC-EVs) naturally and transiently transfer functional P-gp to maternal decidual cells, restoring lost and or reduced P-gp function (exofection) to sustain pregnancy homeostasis. A similar loss of P-gp at the blood brain barrier (BBB) contributes to impaired amyloid-{beta} (A{beta}) clearance and neuroinflammation in Alzheimers disease. We investigated whether CTC-EV-mediated exofection could restore P-gp function in human brain endothelial cells (hBECs) and enhance A{beta} clearance under inflammatory and neurodegenerative conditions. MethodsCTC-EVs were isolated and characterized by nanoparticle tracking analysis and western blotting for P-gp and EV markers. Transcriptomic profiling of CTC-EVs identified enrichment of transporter-related genes, including solute carriers and ABC transporters, along with inflammatory mediators. Network analysis revealed coordinated modules linking EV cargo to transporter regulation, endocytosis/trafficking pathways, and inflammatory remodeling processes converging on BBB efflux activity. hBECs were exposed to LPS (500 ng/mL, 48 h) with or without CTC-EVs. P-gp expression was assessed by immunofluorescence (mean fluorescence intensity, MFI) and western blotting, while functional efflux was measured using Calcein-AM assays. A{beta} oligomer transport was evaluated using a transwell hBEC model. In vivo, 3xTg-AD mice received intravenous CTC-EVs (1x10L/day for 5 days), followed by assessment of P-gp expression, A{beta} burden, and neuroinflammatory markers. Pharmacokinetic studies in P-gp knockout mice were conducted to confirm functional transporter recovery. ResultsLPS exposure significantly reduced P-gp expression in hBECs (41.3% decrease in MFI, p=0.0084), which was restored by CTC-EVs (46.7% increase vs. LPS, p=0.0121). Exofection increased P-gp by a 2.1-fold following EV treatment as determined by western blot. Functional assays demonstrated enhanced efflux, with a 38.5% reduction in intracellular Calcein fluorescence (p<0.001). Network-informed mechanisms supported coordinated regulation of transporter and trafficking pathways. CTC-EVs improved A{beta} transport across inflamed hBEC monolayers. In vivo, EV-treated 3xTg-AD mice exhibited increased P-gp expression in the frontal cortex (38.6%) and hippocampus (42.1%), reduced A{beta} plaque burden (27.9%), and decreased inflammatory markers (IL-1{beta} and TNF-, p<0.05). In P-gp knockout mice, EVs reduced brain drug accumulation by 22.4% (p=0.032), confirming restoration of transporter function. ConclusionCTC derived EVs are natural carriers of functional transporter proteins and restore efflux capacity in compromised endothelial barriers. Integration of transcriptomic and network analyses highlights coordinated regulation of transporter, trafficking, and inflammatory pathways underlying exofection. This reproductive biology inspired strategy offers a promising therapeutic approach for enhancing A{beta} clearance and mitigating neuroinflammation in Alzheimers disease.

Proteins can be actively packaged into extracellular vesicles (EVs) through mechanisms dependent on the stimulus that activated the cells. Identifying proteins released in endothelial EVs in response to stimuli relevant to cardiovascular disease (CVD) may therefore reveal potential biomarkers that provide information about the vascular endothelium. This study aimed to identify differentially expressed proteins in EVs released from human umbilical vein endothelial cells (HUVEC) in response to stimuli relevant to vascular endothelium activation. HUVEC were stimulated with TNF (10 ng/mL) or oxLDL (10 {micro}g/mL). Apoptosis was assessed using a flow cytometric DNA fragmentation protocol and caspase-3/7 activity assay. Size distributions of EVs were examined by nanoparticle tracking analysis. Isolated EVs were examined using tandem liquid-chromatography-mass spectrometry (LC-MS/MS). While treatment of HUVECs with TNF or oxLDL resulted in non-significant elevations in levels of EVs, only TNF increased apoptosis. Mass spectrometry quantified 1355 proteins and revealed significant differences in the proteome of EVs from TNF-treated HUVEC compared to EVs from oxLDL-treated or untreated cells. Several candidate biomarkers were significantly and differentially expressed in response to TNF, including E-selectin and dual specificity phosphatase 7. This study further associated E-selectin on endothelial-derived EVs with endothelial apoptosis and may offer a biomarker of endothelial damage in patients with CVD.

BackgroundSmall extracellular vesicles (EVs) are nanosized, endosome-derived particles which transfer RNA, proteins, and bioactive molecules to mediate intercellular communication. While EV signaling has been observed in many organ systems, it remains unclear whether glomerular endothelial cell (GEC)-derived small EVs directly interact with podocytes in vivo or how they traverse the glomerular basement membrane (GBM). MethodsGEC-derived small EVs were characterized by nanoparticle tracking analysis, electron microscopy, RAMAN spectroscopy and flow cytometry. Cargo composition was analyzed by proteomics, and microRNA (miR) profiling. Functional and structural features were examined using protease, collagenase, adhesion, and multimodal imaging assays. GEC-derived small EV uptake and downstream transcriptional effects were studied in cultured podocytes, while in vivo trafficking was assessed by injection of labeled small EVs into transgenic zebrafish larvae under baseline conditions, puromycin-induced damage, and cd2ap-knockdown. ResultsGECs released bona fide exosome-like small EVs carrying a highly cell type-specific miR cargo. Small EV transfer to podocytes induced a defined transcriptional response consistent with miR-mediated repression of target genes involved in extracellular matrix organization, cell cycle regulation, and cellular stress responses. Proteomic analyses revealed enrichment of surface proteases and integrin-associated proteins that conferred sustained proteolytic activity and enabled GEC-derived small EV migration through extracellular matrix surrogates. In vivo, circulating small EVs traversed the GBM and localized selectively to podocytes in healthy glomeruli, whereas glomerular injury permitted small EV entry into the tubular compartment. ConclusionThese findings provide first in vivo evidence that GEC-derived small EVs can cross the GBM and impact on podocytes. By identifying integrin- and protease-dependent mechanisms which facilitate vesicle passage, this study redefines the GBM as a dynamic interface of heterocellular, vesicle-mediated communication.

Extracellular vesicles (EVs) hold significant promise as biomarkers, but their clinical translation is constrained by variability in pre-analytical handling and isolation. EV isolation methods directly shape which EV populations are captured and characterized, yet systematic method comparisons across multiple analytical dimensions are limited. We comprehensively evaluated eleven EV isolation methods to define their performance and applications. EVs were quantified by NanoFCM, profiled for tetraspanins (CD9, CD63, CD81) via MSD assays, and further characterized by LC-MS/MS proteomics. We show that different EV isolation methods recover different EV populations. Our data provide guidance on method selection based on downstream application needs and serve as a look-up tool if a protein of interest is detected. EV isolation methods broadened proteome coverage but showed divergent performance and recover different EV populations. While all methods captured EVs in the 50-150nm range, centrifugation and ultracentrifugation identified the broadest proteomes (up to 1093 proteins) driven by higher plasma protein carryover. Conversely, ExoEasy and qEV 70 isolated larger EVs and achieved stronger depletion of abundant plasma proteins but showed lower proteome coverage. A total of 117 proteins were detected across all isolation methods. Pre-clearing samples removed contaminants but at the cost of protein identifications. We demonstrate that method selection must align with the specific analytical goal: centrifugation for comprehensive proteome profiling, affinity/size-exclusion methods for contaminant-sensitive assays, and precipitation for high-throughput applications. This systematic characterization provides an evidence-based framework and look-up resource for matching isolation strategies to downstream applications and research questions. Graphical Abstract for Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/710675v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@12ad967org.highwire.dtl.DTLVardef@270e4eorg.highwire.dtl.DTLVardef@1c41bcorg.highwire.dtl.DTLVardef@11fb236_HPS_FORMAT_FIGEXP M_FIG C_FIG This study evaluated 11 extracellular vesicle (EV) isolation methods which enriched distinct EV subpopulations with varying degrees of contaminants. No single approach optimized purity or proteome coverage; in this paper we present an Evidence-Based Framework to select plasma EV isolation methods based on downstream application needs.

Macrophages are present at high frequencies in transplanted organs and solid tumors. These infiltrating macrophages are highly tuned by tissue niche signals. However, the underlying mechanisms that orchestrate the activation of distinct macrophage populations remain unclear. Here, using heart transplantation and tumor implantation models, we found that macrophages in cardiac allografts exhibited higher intracellular iron (Fe2+) level and higher expression of the iron transporter SLC11A1 than those in the solid tumors. In mice, the myeloid-specific deletion of Slc11a1 alleviated the proinflammatory state of macrophages and cardiac allografts rejection. Together, our findings identify SLC11A1 as a new player in immune response regulation, implicating SLC11A1 as a therapeutic target for both allograft and tumor rejection. This preprint reports initial findings; additional experiments are ongoing and will be included in a future full manuscript.

Pathogenic bacterial extracellular vesicles (BEVs) can disrupt the blood-brain barrier (BBB), leading to neuroinflammation. Prior in vitro studies of this process were performed in simple models that may have lacked important physiological factors. We sought to determine if treatment with Escherichia coli-derived BEVs could directly compromise the integrity of a BBB lab-on-chip model or if an immune component was required. Our device featured isogenic human induced pluripotent stem cell-derived brain microvascular endothelial-like cells (BMECs) and pericytes separated by an ultrathin, porous silicon nitride membrane. BEVs and free lipopolysaccharide (LPS) were capable of causing upregulation of intercellular adhesion molecule-1 on the BMEC surfaces, which is important for immune cell recruitment. However, neither BEVs nor LPS at physiological doses caused pronounced loss of BMEC tight junction proteins, nor did they increase barrier permeability to small dye molecules. In contrast, stimulating THP-1 macrophages with BEVs led to increased production of pro-inflammatory cytokines, and conditioned media from the stimulated macrophages disrupted BMEC tight junctions and increased barrier permeability. Our work demonstrates the importance of incorporating an immune component in studies of BEV-mediated disruption of BBB models.