Extracellular Vesicles Concentration Research Articles

(Digital Presentation) A Microfluidic Approach for Direct Immobilization of Extracellular Vesicles on Gold Nanoislands for Efficient Detection Using Localized Surface Plasmon Resonance

Extracellular vesicles (EVs) are crucial for intercellular communication and are potential biomarkers for cancer diagnosis. To improve the detection of EVs, we have developed a platform that uses an ultra-thin film of gold to form gold nanoislands. This platform allows for the direct immobilization of EVs onto the gold nanoislands, using a simple microfluidic device. Detection of the EVs is based on the Localized Surface Plasmon Resonance (LSPR) method. With this innovative approach, we aim to enhance the accuracy and efficiency of EV detection, which could ultimately lead to faster detection and therefore the possibility of an early-stage diagnosis of cancer.Gold nanoparticles are deposited on glass substrates using physical vapor deposition to create an ultra-thin layer of gold. Annealing the nanoparticle film forms gold nano-islands, which are drops with varying shapes and sizes from solid dewetting. A PDMS microfluidic device with a tapered channel of 200-micron depth and a 4mm diameter collection chamber bonded to gold-coated substrates is used to detect EVs from MCF7. The EVs flow through the channel at a rate of 10 µL/min, and the LSPR spectrum of the gold nanoislands is measured before and after EV immobilization. Changes in the refractive index of the surrounding medium due to the presence of EVs cause a shift in the Au LSPR band, which can be used to detect EVs.In this study, we will utilize extracellular vesicles (EVs) obtained from a breast cancer cell line (MCF7) that had been grown in a small bioreactor. A simple microfluidic device will be designed and fabricated to evaluate the Localized Surface Plasmon Resonance (LSPR) shift of the gold plasmon band for different EV concentrations. The microfluidic device was chosen due to its high reproducibility, sensitivity, and ability to work with small amounts of EVs. To understand the sensitivity of this platform, a calibration curve will be plotted and compared with the results of other platforms that we have previously studied. To validate the LSPR detection using the microfluidic device, the eluted exosomes from the device will be characterized using TRPS and FESEM techniques.Overall, our findings will demonstrate the potential of utilizing EVs and LSPR-based microfluidic devices as a promising tool for the detection of EVs and further clinical analysis for cancer diagnosis.

Gold Nanoparticle (GNP) Enhanced Electroporation of Extracellular Vesicles to Promote Gene Loading

Gene therapy is an emerging and powerful therapeutic tool for treating diverse diseases via functional genetic material delivery to targeted defective genes in cells. Extracellular vesicles (EVs) are natural carriers released by all living cells, which are gaining attention for usage in advancing efficiency of therapeutic delivery due to their various advantageous properties, including; (i) cargo protection from enzymatic degradation, (ii) tissue-specific targeting, and (iii) high biocompatibility. EVs can be isolated from a targeted set of cells and used for therapeutic delivery as they can travel back to the same cell type due to their unique surface proteins and markers to release the genes at the specified site. One of the leading methods for cargo transfection into EVs is electroporation, which utilizes short-duration voltage pulses to create pores on the EV surface membrane, during which the cargo can enter. The voltage creates a potential difference between the less electrically conductive EV membrane and the more electrically conductive internal and external EV environments leading to the formation of these pores. This process is limited by the maximum pulse amplitude and duration the EVs can withstand for maximum pore formation and cargo encapsulation while still permitting membrane recovery afterward. We specifically use microfluidic electroporation, which we have previously found to be the most efficient in loading. This technique utilizes water-in-oil droplet encapsulation of the EVs which are then electroporated by voltage application as they pass through parallel metal electrodes. As the droplets pass through the microfluidic device, the non-conductive oil phase creates a barrier allowing for specific consistent and short-pulsed voltage delivery for each EV-encapsulated conductive water droplet [1]. We aim to increase efficiency of gene therapy loading into EVs using gold nanoparticle (AuNP) enhanced electroporation to provide higher concentration, targeted, and protected gene delivery. It has been shown that AuNP can increase the loading into cells during electroporation by allocating the electric voltage onto the cell surface creating a pulse strength focusing effect, allowing more pore formation without increasing the total voltage applied [2]. Due to similar surface properties, we hypothesized that AuNPs would provide a similar increase in transfection of EVs. To test this hypothesis, we electroporated green fluorescence protein (GFP) into EVs with the introduction of AuNPs at various concentrations. Fluorescence analysis through Cytation 5 and EV concentration measurement with Nanoparticle Tracking Analysis (NTA) revealed a close to double increase in the GFP molecules per EV with the AuNP incorporation. Assessment with NTA and Transmission Electron Microscopy showed that the AuNPs did not significantly change the size, concentration, zeta potential, or morphology of the EVs, compared to the native EVs without electroporation, for retaining the EV originality and integrity. Overall, AuNPs have the capacity to increase cargo transfection into EVs, allowing high-efficiency packaging for specific therapy delivery to enhance effectiveness of the treatment.[1] Geng, T., & Lu, C. (2013). Microfluidic electroporation for cellular analysis and delivery. Lab Chip, 13(19), 3803–3821. https://doi.org/10.1039/c3lc50566a[2] Zu, Y., Huang, S., Liao, W.-C., Lu, Y., & Wang, S. (2014). Gold Nanoparticles Enhanced Electroporation for Mammalian Cell Transfection. Journal of Biomedical Nanotechnology, 10(6), 982–992. https://doi.org/10.1166/jbn.2014.1797 Figure 1

From sweat to hope: The role of exercise-induced extracellular vesicles in cancer prevention and treatment.

The benefits of regular physical exercise on cancer prevention, as well as reducing fatigue, treatment side effects and recurrence, and improving quality of life and overall survival of cancer patients, are increasingly recognised. Initial studies showed that the concentration of extracellular vesicles (EVs) increases during physical activity and that EVs carry biologically active cargo. These EVs are released by blood cells, skeletal muscle and other organs involved in exercise, thus suggesting that EVs may mediate tissue crosstalk during exercise. This possibility triggered a great interest in the study of the roles of EVs in systemic adaptation to exercise and in their potential applications in the prevention and treatment of various diseases, including cancer. This review presents studies exploring the concentration and molecular cargo of EVs released during exercise. Furthermore, we discuss putative stimuli that may trigger EV release from various cell types, the biological functions and the impact of exercise-induced EVs on cancer development and progression. Understanding the interplay between exercise, EVs, and cancer biology may offer insights into novel therapeutic strategies and preventive measures for cancer.

Serum Extracellular Vesicles Cargo Approach in Bitches with Mammary Tumors.

This study investigated serum extracellular vesicles (EVs) in bitches with mammary neoplasms, in order to understand their size, shape, and concentration, as well as their association with tumor malignancy. Thirty bitches were categorized into control (n = 10), mammary tumor grades I and II (GI, n = 13), and grade III (GII, n = 7). Serum was separated from blood collected during mastectomy, and EVs were isolated using size exclusion chromatography. The analysis revealed no significant differences in EV concentrations among groups, with similar concentrations for control, GI, and GII. Ninety-one proteins were identified in EV-enriched samples, with six showing varied abundance across groups. Notably, keratin 18 was highly abundant in GI, while sushi domain-containing protein, EvC ciliary subunit 2, and the joining chain of multimeric IgM and IgA were increased in GII. Additionally, protocadherin 17 and albumin were upregulated in both GI and GII. ROC curves identified potential biomarkers for differentiating tumor grades. Enrichment pathway analysis revealed AFP gene upregulation in the GI. Mass spectrometry proteomics data were deposited in Mendeley Data. The study provides valuable insights into serum EV characterization in bitches, suggesting keratin 18 and protocadherin 17 as potential biomarkers for canine mammary neoplasia, with implications for future diagnostic and therapeutic strategies.

Optimizing of a suitable protocol for isolating tissue-derived extracellular vesicles and profiling small RNA patterns in hepatocellular carcinoma.

Extracellular vesicles (EVs) facilitate cell-cell interactions in the tumour microenvironment. However, standard and efficient methods to isolate tumour tissue-derived EVs are lacking, and their biological functions remain elusive. To determine the optimal method for isolating tissue-derived EVs, we compared the characterization and concentration of EVs obtained by three previously reported methods using transmission electron microscopy, nanoparticle tracking analysis, and nanoflow analysis (Nanoflow). Additionally, the differential content of small RNAs, especially tsRNAs, between hepatocellular carcinoma (HCC) and adjacent normal liver tissues (ANLTs)-derived EVs was identified using Arraystar small RNA microarray. The targets of miRNAs and tsRNAs were predicted, and downstream functional analysis was conducted using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, non-negative matrix factorization and survival prediction analysis. A differential centrifugation-based protocol without cell cultivation (NC protocol) yielded higher EV particles and higher levels of CD9+ and CD63+ EVs compared with other isolation protocols. Interestingly, the NC protocol was also effective for isolating frozen tissue-derived EVs that were indistinguishable from fresh tissue. HCC tissues showed significantly higher EV numbers compared with ANLTs. Furthermore, we identified different types of small RNAs in HCC tissue-derived EVs, forming a unique multidimensional intercellular communication landscape that can differentiate between HCC and ANLTs. ROC analysis further showed that the combination of the top 10 upregulated small RNAs achieved better diagnostic performance (AUC = .950 [.895-1.000]). Importantly, most tsRNAs in HCC tissue-derived EVs were downregulated and mitochondria-derived, mainly involving in lipid-related metabolic reprogramming. The NC protocol was optimal for isolating EVs from HCC, especially from frozen tissues. Our study emphasized the different roles of small-RNA in regulating the HCC ecosystem, providing insights into HCC progression and potential therapeutic targets.

Characterisation of LPS+ bacterial extracellular vesicles along the gut-hepatic portal vein-liver axis.

Gut microbiome dysbiosis is a major contributing factor to several pathological conditions. However, the mechanistic understanding of the communication between gut microbiota and extra-intestinal organs remains largely elusive. Extracellular vesicles (EVs), secreted by almost every form of life, including bacteria, could play a critical role in this inter-kingdom crosstalk and are the focus of present study. Here, we present a novel approach for isolating lipopolysaccharide (LPS)+ bacterial extracellular vesicles (bEVLPS) from complex biological samples, including faeces, plasma and the liver from lean and diet-induced obese (DIO) mice. bEVLPS were extensively characterised using nanoparticle tracking analyses, immunogold labelling coupled with transmission electron microscopy, flow cytometry, super-resolution microscopy and 16S sequencing. In liver tissues, the protein expressions of TLR4 and a few macrophage-specific biomarkers were assessed by immunohistochemistry, and the gene expressions of inflammation-related cytokines and their receptors (n=89 genes) were measured using a PCR array. Faecal samples from DIO mice revealed a remarkably lower concentration of total EVs but a significantly higher percentage of LPS+ EVs. Interestingly, DIO faecal bEVLPS showed a higher abundance of Proteobacteria by 16S sequencing. Importantly, in DIO mice, a higher number of total EVs and bEVLPS consistently entered the hepatic portal vein and subsequently reached the liver, associated with increased expression of TLR4, macrophage markers (F4/80, CD86 and CD206), cytokines and receptors (Il1rn, Ccr1, Cxcl10, Il2rg and Ccr2). Furthermore, a portion of bEVLPS escaped liver and entered the peripheral circulation. In conclusion, bEV could be the key mediator orchestrating various well-established biological effects induced by gut bacteria on distant organs.

Differential extracellular vesicle concentration and their biomarker expression of integrin αv/β5, EpCAM, and glypican-1 in pancreatic cancer models

Tumor-derived extracellular vesicles (EVs) show great potential as biomarkers for several diseases, including pancreatic cancer, due to their roles in cancer development and progression. However, the challenge of utilizing EVs as biomarkers lies in their inherent heterogeneity in terms of size and concentration, making accurate quantification difficult, which is highly dependent on the isolation and quantification methods used. In our study, we compared three EV isolation techniques and two EV quantification methods. We observed variations in EV concentration, with approximately 1.5-fold differences depending on the quantification method used. Interestingly, all EV isolation techniques consistently yielded similar EV quantities, overall size distribution, and modal sizes. In contrast, we found a notable increase in total EV amounts in samples from pancreatic cancer cell lines, mouse models, and patient plasma, compared to non-cancerous conditions. Moreover, individual tumor-derived EVs exhibited at least a 3-fold increase in several EV biomarkers. Our data, obtained from EVs isolated using various techniques and quantified through different methods, as well as originating from various pancreatic cancer models, suggests that EV profiling holds promise for the identification of unique and cancer-specific biomarkers in pancreatic cancer.

Higher EpCAM-Positive Extracellular Vesicle Concentration in Ascites Is Associated with Shorter Progression-Free Survival of Patients with Advanced High-Grade Serous Carcinoma.

Platinum-resistant high-grade serous carcinoma (HGSC) is an incurable disease, so biomarkers that could help with timely treatment adjustments and personalized approach are extensively being sought. Tumor-derived extracellular vesicles (EVs) that can be isolated from ascites and blood of HGSC patients are such promising biomarkers. Epithelial cell adhesion molecule (EpCAM) expression is upregulated in most epithelium-derived tumors; however, studies on prognostic value of EpCAM overexpression in ovarian carcinoma have shown contradictory results. The aim of our study was to evaluate the potential of total and EpCAM-positive EVs as prognostic and predictive biomarkers for advanced HGSC. Flow cytometry was used to determine the concentration of total and EpCAM-positive EVs in paired pretreatment ascites and plasma samples of 37 patients with advanced HGSC who underwent different first-line therapy. We found that higher EpCAM-positive EVs concentration in ascites is associated with shorter progression-free survival (PFS) regardless of treatment strategy. We also found a strong correlation of EpCAM-positive EVs concentration between ascites and plasma. Our findings indicate that EpCAM-positive EVs in ascites of patients with advanced HGSC have the potential to serve as prognostic biomarkers for predicting early recurrence and thereby likelihood of more aggressive tumor biology and development of chemoresistance.

Unveiling the complexity: Liquid biopsy-based proteomic exploration of triple-negative breast cancer in women.

e13138 Background: This groundbreaking study places a spotlight on breast cancer in women, with a specific emphasis on the challenging triple-negative breast cancer (TNBC) subtype. Delving into the intricate world of extracellular vesicles (EVs) from TNBC-diagnosed individuals, the research meticulously compares various conditions, including healthy controls, benign tumors, recurrent tumors, and those in remission. Employing cutting-edge techniques like liquid biopsy and LC-MS/MS, the study meticulously explores the proteomic profile of serum exosomes, aiming to pinpoint diagnostic and prognostic markers in breast cancer. Methods: In the initial phase, EVs were expertly isolated and characterized from blood plasma samples exclusively from women, utilizing state-of-the-art methods such as ultracentrifugation and nanoparticle tracking analysis (NTA). Despite applying rigorous statistical criteria, the study found no significant differences in EV concentrations and sizes among the diverse groups. However, the ensuing proteomic analysis uncovered 130 proteins, with Cystatin A and Histone H2A emerging as promising biomarkers for TNBC. To validate them, the study intricately tested tumor cell lines from distinct subtypes, representing luminal, triple-negative, and HER2 overexpressed. The examination of gene and protein expression through RT-qPCR and western blotting added a layer of depth to the research, unraveling the complexity of regulatory processes within cells and demonstrating a remarkable congruence between protein levels found in the proteomics from patients’ serum and in immortalized cell lines. Results: The proteomic analysis brought to light the absence of CSTA and HIST1H2A expression in benign tumor patients, varied levels in malignant tumor patients, and a substantial increase in TNBC patients pre-mastectomy. Validating these findings through western blotting further accentuated the potential of these proteins as distinguishing factors. Conclusions: In conclusion, this pioneering study not only underscores the immense potential of liquid biopsy-based proteomic analysis in identifying crucial diagnostic and prognostic markers in breast cancer but also sheds light on the intricate nuances of the challenging TNBC subtype. Our findings unveil valuable insights that pave the way for future personalized treatment strategies, marking a significant stride forward in breast cancer research.

Enhanced visualization of articular cartilage chondroprogenitor-derived extracellular vesicles using cytospin centrifugation for confocal imaging

Characterizing Extracellular Vesicles (EVs) poses a challenge due to their complex composition and small size. Efforts to visualize fluorescently labeled EVs post-isolation with confocal microscopy encounter a significant obstacle in congregating the isolates on slides. While cytospin is a well-established method for cells, its application with EVs remains unexplored. This study aimed to isolate EVs and evaluate the efficiency of cytospin preparation to enhance their visualization using confocal microscopy. Following informed consent, cartilage shavings from a non-diseased human tibiofemoral joint were digested to obtain cartilage-resident chondroprogenitors through a migratory-assay (MCP). The MCPs were characterized for MSC markers (positive: CD105, CD73, CD90; negative: HLA-DR, CD34, CD45) using FACS. The conditioned medium was utilized to isolate EVs using the cutoff filter and precipitation technique, and their confirmation for transmembrane markers CD63 and CD81 was performed via FACS. Subsequently, the isolated EVs were subjected to confocal microscopy with and without (control) cytospin preparation. The MCPs exhibited high expression of positive and low expression of negative MSC markers. The study effectively employed cytospin centrifugation and confocal imaging to robustly identify EVs isolated from MCPs. Fluorescently labeled with CD63 and CD81 markers, the EVs isolated using cytospin centrifugation were concentrated on glass slides, and compared to control slides; the experimental setup facilitated a 100 times concentration of EVs, thereby enabling easy identification. Confocal analysis confirmed positive expressions of CD63 and CD81, aligning with FACS results. This study introduces a novel approach that enhances the visualization of MCP-derived EVs using cytospin centrifugation.

Extracellular Vesicles to Predict Outcomes After Transcatheter Aortic Valve Implantation – a Prospective, Multicenter Cohort Study

IntroductionTranscatheter aortic valve implantation (TAVI) is an established treatment for aortic stenosis (AS) in patients at intermediate and high surgical risk. Circulating extracellular vesicles (EVs) are nanoparticles involved in cardiovascular diseases. We aimed to (i) determine the effect of TAVI on plasma concentrations of five EV subtypes and (ii) evaluate the predictive value of EVs for post-TAVI outcomes.MethodsBlood samples were collected 1 day before TAVI and at hospital discharge. Concentrations of EVs were evaluated using flow cytometry.ResultsConcentration of leukocytes EVs decreased after TAVI, compared to the measurement before (p = 0.008). Among 123 patients discharged from the hospital, 19.5% experienced MACCE during the median of 10.3 months. Increased pre-TAVI concentration of phosphatidylserine-exposing EVs was an independent predictor of MACCE in multivariable analysis (OR 5.313, 95% CI 1.164–24.258, p = 0.031).ConclusionsPatients with increased pre-TAVI concentration of procoagulant, PS-exposing EVs have over fivefold higher odds of adverse outcomes.Graphical

#1984 Extracellular vesicles drive loss of endothelial homeostasis in pediatric chronic kidney disease

Abstract Background and Aims Cardiovascular disease (CVD) is the main cause of mortality in chronic kidney disease (CKD). However, the pathogenesis of CVD in CKD remains incompletely understood. Extracellular vesicles (EVs) emerged as mediators of inter-organ cross-talk and endothelial EVs (EC-EVs) were previously associated with CVD. We hypothesized that CKD drives endothelial EV release in CKD and that these EC-EVs drive CVD in CKD. Method A cohort of 94 children (mean age 10.9 years) at different stages of CKD (patients with or without dialysis and after kidney transplantation (KTx)) and age-matched healthy donors was recruited, offering the unique opportunity to analyze cardiovascular effects of CKD and EV dynamics in absence of age-related comorbidities. Thorough characterization of plasma EVs by electron microscopy, nanoparticle tracking analysis (NTA), flow cytometry and miRNA sequencing was carried out. Functional properties of patient EVs and CKD-specific EV release mechanisms were tested in vitro. Results EC-EVs were elevated in hemodialysis (HD) patients (as measured by flow cytometry) while total EV counts showed higher concentrations in peritoneal dialysis patients (according to NTA). Longitudinal follow-ups revealed that EC-EVs decreased after receiving KTx. EV small RNA sequencing showed lower abundance of 30 microRNAs in EVs from CKD patients, most pronounced in HD patients compared to both healthy donors and KTx recipients. Downregulation of let-7d-5p, miR-19a-3p, miR-24-3p, miR-103a-3p and miR-142-3p was confirmed using RT-qPCR. Gene enrichment analyses predicted angiogenesis and smooth muscle cell (SMC) proliferation as targets of differentially regulated miRNAs in CKD. In vitro, angiogenesis of Human Umbilical Vein Endothelial Cells (HUVECs) was decreased upon treatment with CKD EVs as indicated by decreased vascular tube formation, endothelial migration and proliferation while SMC proliferation remained unchanged. To assess the putative role of uremic toxins for EV release in CKD, we focused on tryptophan metabolites and performed targeted plasma metabolomics that showed stage-dependent increases of uremic toxins of indole and kynurenine pathways in children with CKD, with highest levels in dialysis patients and almost normal levels after KTx. Uremic toxins indoxyl sulfate, formylkynurenine and xanthurenic acid correlated positively with EC-EVs in our cohort, but were not sufficient to trigger EV release from aortic endothelial cells alone. However, high shear stress together with indoxyl sulfate increased EV release from venous endothelial cells corresponding to higher endothelial EV concentrations in HD patients with arterio-venous fistulas (AVF) and recapitulated EV miRNA changes in vitro observed in CKD in vivo (lower abundance of let-7d-5p and miR-24-3p). Conclusion Endothelial EV release and decreased EV miRNAs associate with advanced CKD while increased endothelial shear stress and indoxyl sulfate form potential causative factors of altered EV phenotype. CKD EVs inhibit angiogenesis in vitro, potentially caused by dysregulated EV miRNAs that could form novel therapeutical targets to reduce CVD burden in CKD.

Effect of the 35 nm and 70 nm Size Exclusion Chromatography (SEC) Column and Plasma Storage Time on Separated Extracellular Vesicles.

The technical difficulty of separating extracellular vesicles (EVs) from plasma proteins in human blood presents a significant hurdle in EV research, particularly during nano ultra-high-performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) analysis, where detecting "vesicular" proteins among abundant plasma proteins is challenging. Standardisation is a pressing issue in EV research, prompting collaborative global efforts to address it. While the MISEV guidelines offer valuable recommendations, unanswered questions remain, particularly regarding sample storage. We compared size exclusion chromatography (SEC) columns with pore sizes of 35 nm and 70 nm to identify fractions with minimal contaminating proteins and the highest concentration of small EVs (sEVs). Following column selection, we explored potential differences in the quality and quantity of sEVs isolated from platelet-free plasma (PFP) after long-term storage at -80 °C (>2.5 years) compared to freshly drawn blood. Our methodologically rigorous study indicates that prolonged storage, under correct storage and processing conditions, does not compromise sEV quality. Both columns effectively isolated vesicles, with the 70 nm column exhibiting a higher abundance of "vesicular" proteins. We propose a relatively rapid and moderately efficient protocol for obtaining a comparatively pure sEV fraction from plasma, facilitating sEV processing in clinical trials.

Poloxamer-188 as a wetting agent for microfluidic resistive pulse sensing measurements of extracellular vesicles.

Microfluidic resistive pulse sensing (MRPS) can determine the concentration and size distribution of extracellular vesicles (EVs) by measuring the electrical resistance of single EVs passing through a pore. To ensure that the sample flows through the pore, the sample needs to contain a wetting agent, such as bovine serum albumin (BSA). BSA leaves EVs intact but occasionally results in unstable MRPS measurements. Here, we aim to find a new wetting agent by evaluating Poloxamer-188 and Tween-20. An EV test sample was prepared using an outdated erythrocyte blood bank concentrate. The EV test sample was diluted in Dulbecco's phosphate-buffered saline (DPBS) or DPBS containing 0.10% BSA (w/v), 0.050% Poloxamer-188 (v/v) or 1.00% Tween-20 (v/v). The effect of the wetting agents on the concentration and size distribution of EVs was determined by flow cytometry. To evaluate the precision of sample volume determination with MRPS, the interquartile range (IQR) of the particles transit time through the pore was examined. To validate that DPBS containing Poloxamer-188 yields reliable MRPS measurements, the repeatability of MRPS in measuring blood plasma samples was examined. Flow cytometry results show that the size distribution of EVs in Tween 20, in contrast to Poloxamer-188, differs from the control measurements (DPBS and DPBS containing BSA). MRPS results show that Poloxamer-188 improves the precision of sample volume determination compared to BSA and Tween-20, because the IQR of the transit time of EVs in the test sample is 11 μs, which is lower than 56 μs for BSA and 16 μs for Tween-20. Furthermore, the IQR of the transit time of particles in blood samples with Poloxamer-188 are 14, 16, and 14 μs, which confirms the reliability of MRPS measurements. The solution of 0.050% Poloxamer-188 in DPBS does not lyse EVs and results in repeatable and unimpeded MRPS measurements.

Abstract 2033: Bone Marrow-Derived Macrophage Extracellular Vesicles From Diabetic Mice Suppress Angiogenic Properties In Endothelial Cells

Introduction: Peripheral artery disease (PAD), increased in diabetes, leads to mortality and disability. Diabetes promotes a proinflammatory macrophage phenotype that suppresses repair. Macrophage-derived extracellular vesicles (EVs) have been associated with tissue damage. We hypothesize that diabetes perturbs macrophage EV cargo, thereby contributing to impaired repair responses to PAD/ischemia. Methods: Unilateral ligation of the left femoral artery (FAL) was performed to induce hind limb ischemia (HLI) in diabetic Akita (C57BL/6-Ins2 Akita /J) vs. non-diabetic (NDM, C57BL/6J) male mice. Murine BMDMs were grown in Exo-free medium. Ultracentrifugation was used to isolate EVs. Western blotting, transmission electron microscopy (TEM) and ZetaView nanoparticle tracking analysis were used to characterize EV size, number, concentration, and morphology. We profiled EV microRNA (miRNA) and protein cargos from Akita vs. NDM BMDM mice. Results: BMDMs-EV size was similar in Akita and NDM mice (diameter, 20-200nm). TEM showed cup-shaped morphology. Western blot confirmed known positive and negative control exosomal markers. Comparing Akita vs. NDM BMDM EVs, a total of 3277 proteins were identified; 821 proteins were significantly differentially expressed. Enrichment analysis highlighted pathways predominantly related to oxidative stress, insulin signaling, actin cytoskeleton, CLEAR, lipid metabolism, phagosome maturation, RNA binding and DNA replication. A total of 912 EV miRNAs were identified; 39 were significantly up-regulated and 19 were down-regulated. Preliminary analysis links a number of them to endothelial function and angiogenesis. In vitro , endothelial tube formation was impaired in human iliac artery endothelial cells treated with diabetic vs. NDM BMDM EVs. BMDM treated with diabetic vs. WT EVs displayed increased expression of inflammatory genes (TNF, IL-6,) and reduced expression of mitochondrial complex I and complex II proteins. Conclusion: We identified microRNAs and distinct proteins from diabetic EVs as potential mediators of impaired endothelial function and resolution of inflammation. Targeting macrophage EVs composition might identify therapeutic approaches to prevent and treat PAD in diabetes.

Skeletal Muscle Extracellular Vesicles Enhance Mitochondrial Movement and have a Dose-dependent Effect on Increasing Mitochondrial Respiration

Chronic exercise leads to systemic health benefits across tissues, including an increase in mitochondrial function in skeletal muscle. One potential mechanism is via the release of extracellular vesicles (EVs), which can transfer functional cargo to recipient cells. Previously, we have shown that using an in vitro model of exercise, chronic contractile activity (CCA)-derived EVs (CCA-EV) increased mitochondrial biogenesis in healthy myoblasts. Here, we hypothesize that CCA-EVs will have a concentration-dependent effect on mitochondrial respiration, and also regulate mitochondrial dynamics in myoblasts. C2C12 myoblasts were differentiated into myotubes, and electrically paced (3hrs/day, 4 days, 14V, C-PACE EM, IonOptix). EVs from control and CCA-stimulated myotubes were isolated from conditioned media using differential ultracentrifugation and biophysically characterized by size and concentration using TRPS (Izon). C2C12 myoblasts were treated daily for 4 days using three different concentrations: 1.0E4; 2.0E4 and 4.0E4 EVs/cell (N=4-5). Basal and maximal oxygen consumption rates (OCR) were measured using the Seahorse XFe24 (Agilent). We used live-cell fluorescence microscopy and Mitometer software to access mitochondrial movement: area/volume, displacement, distance, length, perimeter, speed and velocity. Average size was unchanged between control-EVs (123±10.8 nm) and CCA-EVs (123±11.9 nm) (p=0.9137, N=3), and 2.3-fold more CCA-EVs were released vs. control-EVs (p=0.2072, N=3). Lowest EV concentration (1.0E+4 EVs/cell, N=5) had no effect on basal (p=0.4226) and maximal (p=0.1835) OCR. Intermediate dosage of CCA-EVs (2.0E+4 EVs/cell, N=5) increased basal OCR by 20% (p=0.0037) and maximal OCR by 18% (p=0.0095), while the highest concentration (4.0E+4 EVs/cell, N=4) decreased basal OCR by 9% (p=0.0001) and maximal OCR by 29% (p=0.0009), compared to control-EVs. Mitochondrial motility (N=484-976) in myoblasts treated daily after each day of CCA showed enhanced mitochondrial dynamics with CCA-EVs: increased area/volume (p<0.0001), displacement (p<0.0001), distance (p<0.0001), mean intensity (p<0.0001), perimeter/surface area (p=0.0137) and speed (p<0.0001), compared to control-EVs. In summary, CCA-EVs induced concentration-dependent improvements in mitochondrial respiration in healthy myoblasts, concomitant with enhanced mitochondrial movement. These results open exciting avenues for the therapeutic potential of CCA-EVs in rescuing mitochondrial dysfunction. TFGS is funded by a Research Manitoba Postdoctoral Fellowship. Grants from CHRIM, CHF, DREAM and NSERC to AS funded the research. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Extracellular vesicles characterization in patients with hypertrophic cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) is diagnosed according to the presence of morphological and functional traits of the heart, often in the presence of genetic mutations. A specific biomarker assessing the aetiopathology of this condition is lacking. Extracellular vesicles (EVs), small particles released by all cells into biological fluids, hold promise as diagnostic and prognostic tools for cardiac diseases. We aim at characterising plasma-derived EVs isolated from 18 consecutive HCM patients and 13 healthy volunteers (CTR). Methods: HCM underwent echocardiographic assessment and genetic testing evaluating 200 genes (NGS). EVs were isolated via ultracentrifugation from platelet-free plasma. Quantitative and qualitative assessments of EVs were performed by nanoparticle tracking analysis and transmission electron microscopy. FACS analysis was used to characterize EV subpopulations. Data are expressed as median and interquartile ranges. Results: Most patients were male (70.8% HCM and 54% CTR) with a median age of 61 (52.5-71) years (HCM) and 47 (44-52) (CTR). Missense mutations in the MYH7 gene were the most found in HCM. The median maximum wall thickness in HCM was 16 mm (15-19) vs 8 mm (7-9) in CTR. No differences were found in EV concentrations between HCM and CTR, respectively, 3.6*109 EV/ml/cell count (2*109-5*109) and 5*10⁹ EV/ml/cell count (4*109-6*109). However, EV concentration was positively associated with the sudden cardiac death risk score in HCM (r= 0.63). Among the EVs positive for CFSE (a specific dye for EVs), those released from platelets, progenitor endothelial cells and neutrophils were increased in HCM patients vs CTR, respectively, by 1.7-, 1.5- and 1.1-fold. A strong negative association (r= -0.74) was found between progenitor endothelial cell-derived EVs and the E/E’ ratio of diastolic function, a strong predictor of first cardiac events. Conclusions: HCM patients present a peculiar phenotypic pattern of EVs that associates which diastolic function and sudden cardiac death.

Blood brain barrier permeability and astrocyte-derived extracellular vesicles in children with juvenile idiopathic arthritis: a cross-sectional study

BackgroundJuvenile idiopathic arthritis (JIA) is the most prevalent rheumatic disease in children, and the inflammatory process is widely studied, primarily characterized by its impact on joint health. Emerging evidence suggests that JIA may also affect the central nervous system (CNS). This study investigates the potential CNS involvement in JIA by analyzing the presence of astrocyte-derived extracellular vesicles (EVs) and the S100B protein in plasma, both of which are indicative of astrocyte activity and blood-brain barrier (BBB) integrity.MethodsEDTA plasma from 90 children diagnosed with JIA and 10 healthy controls, matched by age and gender, was analyzed for extracellular vesicles by flow cytometric measurement. Astrocyte-derived EVs were identified using flow cytometry with markers for aquaporin 4 (AQP-4) and glial fibrillary acidic protein (GFAP). Levels of the S100B protein were measured using a commercial ELISA. Disease activity was assessed using the Juvenile Arthritis Disease Activity Score (JADAS27, 0–57), and pain levels were measured using a visual analogue scale (VAS, 0–10 cm).ResultsOur analyses revealed a significantly higher concentration of astrocyte-derived EVs in the plasma of children with JIA compared with healthy controls. Furthermore, children with JADAS27 scores of 1 or higher exhibited notably higher levels of these EVs. The S100B protein was detectable exclusively in the JIA group.ConclusionThe elevated levels of astrocyte-derived EVs and the presence of S100B in children with JIA provide evidence of BBB disruption and CNS involvement, particularly in those with higher disease activity. These findings underscore the importance of considering CNS health in the comprehensive management of JIA. Further research is required to elucidate the mechanisms behind CNS engagement in JIA and to develop treatments that address both joint and CNS manifestations of the disease.

Extracellular vesicles secreted by 3D tumor organoids are enriched for immune regulatory signaling biomolecules compared to conventional 2D glioblastoma cell systems.

Newer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterize extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems. Primary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterization was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analyzed by high-resolution mass spectrometry. We observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR-323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer-associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response. Our results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.

Characteristics of the Follicular Fluid Extracellular Vesicle Molecular Profile in Women in Different Age Groups in ART Programs.

The aim of this study was to investigate the molecular composition of follicular fluid (FF) extracellular vesicles (EVs) in women of different reproductive ages and its possible relationship to sperm fertilizing ability. FF EVs were obtained by differential centrifugation. The concentration and size distribution of FF EVs were analyzed by nanoparticle tracking analysis. The lipidome and proteome were analyzed by liquid chromatography-mass spectrometry. The isolated FF EVs had a variety of shapes and sizes; their concentration and size distribution did not differ significantly between the age groups. In women younger than 35 years, the concentration of vesicular progesterone was 6.6 times higher than in women older than 35 years, and the total levels of the main lipid classes were increased in younger women. A proteomic analysis revealed that not only FF EV-specific proteins, but also proteins involved in sperm activation were present. New data were obtained on the composition of FF EVs, confirming their importance as molecular indicators of age-related changes in the female reproductive system. In addition, these results shed light on the possible interaction between the FF EVs of women in different age groups and male germ cells. Therefore, studying the transcriptomic and metabolomic profile of FF EVs may be a crucial approach to evaluate the efficacy of ART.