Extracellular Vesicles Isolation Research Articles

Optimization of plasma extracellular vesicle enrichment for in‐depth lipidomic profiling in Alzheimer’s disease

AbstractBackgroundLipid dyshomeostasis is associated with various disorders including Alzheimer’s disease (AD). The work from our laboratory (Su et al, JEV 2021), and others, suggests that extracellular vesicles (EVs) contain lipid biomarkers that could aid in the diagnosis of AD and diseases associated with impaired lipid metabolism.Knowing the lipid content of EVs is a key first step to discovering EV‐based lipid biomarkers in blood. Some studies have reported the lipid content of ‘’EVs’’ in blood. However, used EV isolation techniques that are known to co‐isolate free lipid and lipoproteins.Here we: 1) demonstrate the importance of size exclusion resin size to isolating highly enriched EVs for lipidomic analysis, 2) have developed a reference EV plasma lipidome for the research community and 3) determining if plasma EVs report on lipid dyshomeostasis in Alzheimer’s disease.MethodHuman plasma EVs were isolated using an assortment of commercially available kits or by density fractionation and size exclusion chromatography and characterised by western blot, transmission electron microscopy, and quantitative mass spectrometry based proteomic and lipidome analysis.ResultOf the high‐throughput EV isolation kits tested, the majority were unable to separate lipoproteins from EVs for the purposes of EV lipidomics. We then relied on comparing labor intensive methods (density gradient and size exclusion) to identify a combination of techniques that isolate the analytes of interest, EV proteins and lipids. Quantitative proteome and lipidome analysis strategies are being applied to highly enriched EVs with the goal of providing the reference lipidome of EVs in plasma and in Alzheimer’s disease.ConclusionTo enhance the confidence of EV lipid identification and reveal the lipidome of EVs in plasma, our study compared and identified techniques to enrich EVs and deplete non‐EV associated lipid and protein from human plasma. These findings serve as a reference resource and the foundation for research investigating plasma EV lipids as Alzheimer’s disease biomarkers.

An innovative charge‐based extracellular vesicle isolation method for highly efficient extraction of EV‐miRNAs from liquid samples: miRQuick

AbstractExtracellular vesicle‐derived microRNAs (EV‐miRNAs) are promising biomarkers for early cancer diagnosis. However, existing EV‐miRNA extraction technologies have a complex two‐step process that results in low extraction efficiency and inconsistent results. This study aimed to develop and evaluate a new single‐step extraction method, called miRQuick, for efficient and high‐recovery extraction of EV‐miRNAs from samples. The miRQuick method involves adding positively charged substances to the sample, causing negatively charged EVs to quickly aggregate and precipitate. A membrane lysate is then added to extract only miRNA. The entire process can be completed within an hour using standard laboratory equipment. We validated the miRQuick method using various analytical techniques and compared its performance to other methods for plasma, urine and saliva samples. The miRQuick method demonstrated significantly higher performance than other methods, not only for blood plasma but also for urine and saliva samples. Furthermore, we successfully extracted and detected nine biomarker candidate miRNAs in the plasma of breast cancer patients using miRQuick. Our results demonstrate that miRQuick is a rapid and efficient method for EV‐miRNA extraction with excellent repeatability, making it suitable for various applications including cancer diagnosis.

Brain extracellular vesicles from Alzheimer’s Disease and Down Syndrome patients mediate spread of senescence‐like phenotype in vitro

AbstractBackgroundAlzheimer’s disease (AD) is the prevalent cause of dementia in older adults. Down Syndrome (DS) is the most common chromosomal disorder with strong predisposition to the development of AD. Cellular senescence contributes in the pathophysiology of both AD and DS but the mechanisms are unclear. Senescence associated secretory phenotype (SASP), which refers to a complex of secreted mediators, including soluble factors and extracellular vesicles (EVs) may contribute to the spread of senescence to bystander cells but its role in neurodegenerative disorders has not been evaluated.MethodParietal cortex tissue from patients with AD, DS, and normal/low‐pathology (NL) cases was obtained from the UC Irvine ADRC. The cryopreserved human tissue was used for small EV isolation by ultracentrifugation and size exclusive chromatography (SEC). Purity of the EV fractions was confirmed by immunoblotting against positive (CD63, CD9, Syntenin‐1) and negative (calnexin and GM130) EV/exosomal markers, transmission electron microscopy, and nanoparticle tracking analysis. Cell‐type specific EV fractions were isolated by immunoprecipitation (IP). Senescence transfer potential of brain‐derived EVs were assessed in neuroblastoma and glioma cells using senescence associate beta‐galactosidase activity assay, and confirmed by other senescence markers.ResultOur preliminary data demonstrated the ability of AD and DS, but not NL samples, to transfer senescence to neuroblastoma SH‐SY5Y and glioma U87‐MG cells. Senescence associate beta‐galactosidase activity was on average around 15% higher in cells treated with AD brain derived EVs (AD‐EVs) compared to NL brain EVs (NL‐EVs). This data correlates with around 20% elevation in p21 expression in AD‐EV treated cells. When senescence transfer potentials were compared between AD‐EVs and DS brain derived EVs (DS‐EVs), DS‐EVs exceeded AD‐EVs in their ability to transfer senescence‐like phenotype with around 40% increase in senescence associate beta‐galactosidase activity in cells treated with DS‐EVs. We are currently evaluating levels of senescence‐associated and SASP markers in AD and DS cells and cell‐type specific brain derived small EVs.ConclusionWe demonstrated an ability of AD and DS brain derived EVs to transfer senescence‐like phenotype in vitro. This phenomenon deserves further investigation to estimate its impact on the progression of AD in Down and non‐Down population along with evaluation its therapeutic potential.

Isolation of spontaneously‐released brain extracellular vesicles: implications for brain pathology

AbstractBackgroundIn the era of Precision Medicine, extracellular vesicles (EVs) exhibit great potential for the theragnostic of brain disorders such as Alzheimer’s disease (AD), a complex disease with no effective treatment and poorly understood risk factors, where pathological heterogeneity and diverse clinical presentations complicate the development of precise patient‐tailored therapies. Thus, the collection and characterization of physiologically relevant EVs, as well as the study of its precipitating/risk factors, are of the utmost importance. However, standard brain EV isolation approaches rely on tissue dissociation, which can contaminate EV preparations with intracellular vesicles.MethodBased on a multiscale analysis, including cryo‐EM, label‐free proteomics, and ExoView, we hereby present a novel purification method of small EVs (sEVs) based on the spontaneous release of EVs from the brain.ResultOur advanced EVs analysis demonstrated that our method represents an efficient purification method that captures a small EV‐enriched population spontaneously released by mouse and human brain tissue. In addition, we tested the significance of the release method under conditions where biogenesis/secretion of sEVs was pharmacologically manipulated and under exposure to chronic stress, a clinically‐relevant precipitant of AD. Here we found that the released method monitors the drug‐evoked inhibition or enhancement of sEVs secretion in a very sensitive manner, while chronic stress induces the secretion of brain exosomes accompanied by memory loss and mood deficits suggesting a potential role of sEVs in the brain response to stress and related stress‐driven brain pathologies.ConclusionOverall, this method will contribute to the characterization of the biomarker profile of physiologically relevant brain‐derived sEVs in brain function and pathology.

Abstract C004: Liquid biopsy based on the lipid profiles in plasma-derived extracellular vesicles which can be utilized for breast cancer diagnosis

Abstract Lipids are a major component of cells that can influence multiple cellular functions. Our group previously found that the breast cancer cells could dramatically change their lipid profile during their progression. Thus, we hypothesized that the lipid profile of extracellular vesicles (EVs) could be used as a biomarker for breast cancer (BC) since the lipid profiles of breast cancer cells can be reflected in that of EVs secreted from cancer cells. In the present study, we optimized a method to isolate EVs from human plasma samples that could be used for the comprehensive lipid analysis. We then explored specific lipid profiles in plasma-derived EVs that could discriminate BC and healthy individuals, tumor subtypes and stages. Plasma-derived EVs from breast cancer (n = 105), benign (n = 11), and healthy individuals (n = 43) were used for lipid analysis. The results showed that the change in proportion of the lipids in the EVs could well discriminate stages and subtypes of BC. The proportion of major phospholipids, phosphatidylethanolamines, phosphatidylserines, phosphatidylcholines (PCs) and phosphatidylinositols (PIs) showed different patterns at different stages. Importantly, PI (18:0/20:3), which we previously identified as a lipid biomarker of disease progression expressed in the primary breast cancer tissue, showed a similar trend in the plasma-derived EVs. This finding supports our initial hypothesis. Besides the phospholipids, sphingomyelins were found to be higher in stage IV. With regard to tumor subtypes, PCs and ceramides were enriched in HER2-positive subtype, while lysoPCs and polyunsaturated triglycerides were enriched in triple negative subtype. Furthermore, a BC diagnostic model consisting of three lipids was successfully established and the model showed good performance in discriminating between healthy individuals and BC in the two independent data sets (the area under the ROC curve was estimated around 0.759 to 0.804). In conclusion, profiling of lipids in plasma-derived EVs has the potential to be used as a liquid biopsy that can diagnose BC, and its stages and subtypes. Further validation and improvement in EV isolation technique would be needed to achieve sufficient performance sufficient for clinical application. Citation Format: Masahiro Kawashima. Liquid biopsy based on the lipid profiles in plasma-derived extracellular vesicles which can be utilized for breast cancer diagnosis [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C004.

Abstract LB_B21: Extracellular RNA signatures of mutant KRAS-driven lung cancer

Abstract RAS genes are the most frequently mutated oncogenes in cancer, yet the effects of oncogenic RAS signaling on the noncoding transcriptome remain unclear. We analyzed the transcriptomes of human airway and bronchial epithelial cells transformed with mutant KRAS to define the landscape of KRAS-regulated noncoding RNAs. We find that oncogenic KRAS signaling upregulates noncoding transcripts throughout the genome, many of which arise from transposable elements (TEs). These TE RNAs exhibit differential expression and are regulated by KRAB zinc-finger (KZNF) genes, which are broadly downregulated in mutant KRAS cells and lung adenocarcinomas in vivo. Using both affinity- and nanofiltration-based EV isolation approaches, we show that specific TE RNAs, lncRNAs, and mRNAs, some of which are prognostic for lung adenocarcinoma (LUAD) patient survival, are preferentially released in extracellular vesicles (EVs). Moreover, we find that inhibition of oncogenic KRAS signaling broadly reprograms the noncoding transcriptome, as evidenced by a substantial increase in TE RNA secretion. Mutant KRAS inhibition also increases the abundance of secreted lncRNAs and retained intron-containing transcripts, while decreasing the mRNA content of EVs. Notably, oncogenic KRAS signaling is required for the secretion of mRNAs from a set of 20 genes that are significantly associated with unfavorable clinical outcomes in LUAD. Our results indicate that mutant KRAS remodels the repetitive noncoding transcriptome, demonstrating the broad scope of intracellular and extracellular RNAs regulated by this oncogenic signaling pathway. Our study also suggests that both coding and noncoding RNAs that are secreted in EVs may serve as mutant KRAS-specific signatures for diagnosing lung cancer via minimally invasive RNA liquid biopsy technology, which we are currently developing as a companion diagnostic for mutant KRAS-targeting therapies. Citation Format: Sree Lakshmi Velandi Maroli, Roman E Reggiardo, Reem Khojah, Daniel H Kim. Extracellular RNA signatures of mutant KRAS-driven lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B21.

A Comparative Analysis of the Protein Cargo of Extracellular Vesicles from Helminth Parasites.

Helminth parasites cause debilitating-sometimes fatal-diseases in humans and animals. Despite their impact on global health, mechanisms underlying host-parasite interactions are still poorly understood. One such mechanism involves the exchange of extracellular vesicles (EVs), which are membrane-enclosed subcellular nanoparticles. To date, EV secretion has been studied in helminth parasites, including EV protein content. However, information is highly heterogeneous, since it was generated in multiple species, using varied protocols for EV isolation and data analysis. Here, we compared the protein cargo of helminth EVs to identify common markers for each taxon. For this, we integrated published proteomic data and performed a comparative analysis through an orthology approach. Overall, only three proteins were common in the EVs of the seven analyzed species. Additionally, varied repertoires of proteins with moonlighting activity, vaccine antigens, canonical and non-canonical proteins related to EV biogenesis, taxon-specific proteins of unknown function and RNA-binding proteins were observed in platyhelminth and nematode EVs. Despite the lack of consensus on EV isolation protocols and protein annotation, several proteins were shown to be consistently detected in EV preparations from organisms at different taxa levels, providing a starting point for a selective biochemical characterization.

Identifying Novel Coagulation Biomarkers for Thrombosis in Patients with Lymphoma

CONCLUSION Thromboembolic events are a common complication of lymphoma, influenced by a number of factors, including status of the disease, patient characteristics and treatment modalities. Recent studies demonstrated presence of hemostatic dysregulation in lymphoma patients with the possible involvement of tissue factor (TF) in the development of thrombotic events; however, more data are needed to link coagulation biomarkers' levels with clinical data and increased thrombotic risk. TF initiates blood coagulation and expressed by nonvascular cells plays a major role in hemostasis, while TF expressed by vascular cells induces intravascular thrombosis. In this study, we aimed to profile coagulation biomarkers and determine the roles of neutrophils, monocytes, platelets and endothelial cells and their interactions in thrombus formation in patients with lymphoma. Patients with newly diagnosed lymphoma were prospectively recruited and followed for all thromboembolic events from the time of diagnosis until 3 months after the last cycle of therapy. Peripheral blood samples were obtained before hematological treatment initiation from 127 patients with lymphoma. The coagulation factors profile (Fibrinogen, P-selectin, FVIII, Thrombin and TF) was measured by enzyme-linked immunosorbent assay (ELISA), while TF expression and monocyte-platelet aggregates were determined by flow cytometry. The isolation, quantification and flow cytometry profiling of extracellular vesicles (EVs) ≤1.0 µm have been done according to the protocol developed by Institute for Medical Research, Belgrade, Serbia. Thromboembolic events were diagnosed objectively based on clinical examination, radiographic studies and laboratory evaluation. The mean patients' age was 55±16 years (range, 18-86 years); 44.9% were males. Most patients (58.2%) had advanced stage disease (III/IV), and 35.4% had ECOG performance status ≥1. More than half of the patients had aggressive forms of lymphoma (64.6%), mostly diffuse large B-cell lymphoma (DLBCL). Thrombosis occurred in 14.3% of patients with lymphoma (18/127), predominantly in patients with aggressive (18.3%) vs. indolent forms (6.8%). Total number of thrombosis was 22 (4 patients had more than one thrombosis site), and venous thrombosis was the most prevalent (72.7%). ThroLy score ≥2 had 72.2% of patients with thrombosis, in contrast to 37% of patients without thrombosis. The median value of TF was higher in patients with thrombosis compared to those without thrombosis (224.41 [93.1-446.0] (median [IQR]) vs. 136.43 [109.4-193.4], p=0.036). The concentration of the other assessed coagulation related cells and biomarkers were not significantly different between patients who developed thrombosis during follow-up and those who did not. In multivariate regression analysis, ThroLy score ≥2 (OR=5.505, 95%CI 1.505‒20.100, p=0.01), TF (OR=1.005, 95%CI 1.002‒1.009, p=0.003), and aggressive form of lymphoma (OR=4.680, 95%CI 1.132‒19.356, p=0.033), were independent prognostic factors for thrombosis occurrence. CD11b +CD16 + neutrophils were increased 8-fold in aggressive (DLBCL) compared to indolent form of lymphoma (p<0.001), as were 2-fold CD61 + platelets and 3-fold CD31 + endothelial cells labelled with TF (CD142 +, p<0.05) procoagulant EVs. The monocyte-platelet aggregates (CD14 +CD45 +CD41 +) were increased 5-fold in DLBCL patients compared to the healthy controls (p<0.001). The presented results support the use of ThroLy score for differentiating the sub-groups of lymphoma patients who are at increased risk for thrombotic events, while profiling coagulation biomarkers in different lymphoma populations may aid in understanding the pathophysiologic mechanisms underlying the thromboembolic events. Special emphasis should be placed on the assessment of TF which might be used as a coagulation biomarker in addition to clinical prediction scores.

Multivalent DNA Flowers for High-Performance Isolation, Detection, and Release of Tumor-Derived Extracellular Vesicles.

Tumor-derived extracellular vesicles (T-EVs) hold great promise for understanding cancer biology and improving cancer diagnostics and therapeutics. Herein, we developed multivalent DNA flowers (DFs) containing repeated and equidistant EpCAM aptamers for the efficient isolation of T-EVs. The multivalent aptamer chains in DFs had good flexibility to adapt to the surface morphology of T-EVs and achieved multivalent ligand-receptor interactions, thus showing enhanced isolation ability compared to monovalent aptamers. Compared with other materials for isolation of EVs, DFs were generated by rolling circle amplification (RCA) and self-assembled into microspheres in a one-pot reaction, and the recognition molecules (aptamers) were directly replicated and assembled during the RCA reaction instead of chemical modification and immobilization on the surface of solid materials. Moreover, as optically transparent biomaterials, the content of EpCAM+ EVs could be directly reflected via membrane-based hydrophobic assembly of signaling modules in DFs@EpCAM+ EVs complex, and we found that the amount of EpCAM+ EVs showed greater accuracy in cancer diagnosis than total EVs (88.3 vs 69.7%) and was also higher than the clinically commonly used marker carcinoembryonic antigen (CEA) (88.3 vs 76.7%). In addition, T-EVs could be released by lysis of DFs with the nuclease, gently and easily, keeping high intact and activity of EVs for downstream biological function studies. These results demonstrated that DFs are efficient and nondestructive tools for isolation, detection, and release of T-EVs.

Exploring capabilities of elemental mass spectrometry for determination of metal and biomolecules in extracellular vesicles

Extracellular vesicles (EVs) are increasingly recognized as crucial components influencing various pathophysiological processes, such as cellular homeostasis, cancer progression, and neurological disease. However, the lack of standardized methods for EV isolation and classification, coupled with ambiguity in biochemical markers associated with EV subtypes, remains a major challenge. This Trends article highlights the most common approaches for EV isolation and characterization, along with recent applications of elemental mass spectrometry (MS) to analyse metals and biomolecules in EVs obtained from biofluids or in vitro cellular models. Considering the promising capabilities of elemental MS, the article also looks ahead to the potential analysis of EVs at the single-vesicle and single-cell levels using ICP-MS. These approaches may offer valuable insights into individual characteristics of EVs and their functions, contributing to a deeper understanding of their role in various biological processes.Graphical

First insight into extracellular vesicle-miRNA characterization in a sheep in vitro model of inflammation

Extracellular vesicles (EVs) and their microRNA (miRNA) cargoes have garnered attention in the veterinary field for their regulatory role in various biological processes. This study aimed to (i) evaluate two techniques of EV isolation from sheep peripheral blood mononuclear cell (PBMC) supernatants using the ultracentrifugation (UC) and reagent (REA) methods and (ii) characterize the EV-miRNA profiles after an in vitro inflammatory environment mediated by lipopolysaccharides (LPS). Sheep peripheral blood was collected, and PBMCs were separated using a density gradient reagent. Subsequently, PBMCs were cultured at 37°C for 24 h (5% CO2), and the supernatants were collected to perform the EV isolation. The presence of CD81+ extracellular vesicle marker was determined, and the purity of isolated EVs was calculated as a ratio between the number of isolated EVs and the protein concentration. Moreover, the morphological characterization revealed mainly round-shaped structures with average sizes of 211 nm for EVs isolated by the UC method and 99 nm for EVs isolated by the REA method. Illumina NextSeq sequencing in a single-end mode was used to characterize the miRNA profile, and the differentially expressed (DE) miRNAs were analyzed using a combination of bioinformatics tools. The results revealed that the REA method is reliable for EV isolation from sheep supernatants. It was considered an improvement of the recovery rate and purity of EVs with the enhancement of the number and the expression levels of characterized miRNAs. The EVs isolated by the UC method after an LPS challenge showed 11 DE miRNAs, among which eight miRNAs were upregulated and three were downregulated. On the other hand, the REA method revealed an EV cargo in which eight DE miRNAs were upregulated and 21 DE miRNAs were downregulated. The master miRNA regulators of the biological process were identified by performing the MIRNA-mRNA network analysis, showing that, among the higher representative miRNAs based on the centrality and betweenness, the miR-26a-5p could have a crucial role in the resolution of inflammation. Moreover, the identification of the let-7 miRNA family in all the EVs showed potential targeted genes that regulate the inflammation and immune responses.

Ultrasensitive and High-Resolution Protein Spatially Decoding Framework for Tumor Extracellular Vesicles.

Proteins localized on the surface or within the lumen of tumor-derived extracellular vesicles (EVs) play distinct roles in cancer progression. However, quantifying both populations of proteins within EVs has been hampered due to the limited sensitivity of the existing protein detection methods and inefficient EV isolation techniques. In this study, the eSimoa framework, an innovative approach enabling spatial decoding of EV protein biomarkers with unmatched sensitivity and specificity is presented. Using the luminal eSimoa pipeline, the absolute concentration of luminal RAS or KRASG12D proteins is released and measured, uncovering their prevalence in pancreatic tumor-derived EVs. The pulldown eSimoa pipeline measured absolute protein concentrations from low-abundance EV subpopulations. The eSimoa assays detected EVs in both PBS and plasma samples, confirming their applicability across diverse clinical sample types. Overall, the eSimoa framework offers a valuable tool to (1) detect EVs at concentrations as low as 105 EVmL-1 in plasma, (2) quantify absolute EV protein concentrations as low as fM, and (3) decode the spatial distribution of EV proteins. This study highlights the potential of eSimoa in identifying disease-specific EV protein biomarkers in clinical samples with minimal pre-purification, thereby driving advancements in clinical translation.

Isolation and Proteomic Analysis of Extracellular Vesicles from Lactobacillus salivarius SNK-6.

The proteins carried by the extracellular vesicles of Lactobacillus salivarius SNK-6 (LsEVs) were identified to provide a foundation for further explorations of the probiotic activities of L. salivarius SNK-6. LsEVs were isolated from the culture media of L. salivarius SNK-6 and morphological analysis was conducted by scanning electron microscopy. Subsequent transmission electron microscopy and nanoparticle tracking analysis were performed to assess the morphology and particle size of the LsEVs. In addition, the protein composition of LsEVs was analyzed using silver staining and protein mass spectrometry. Finally, internalization of the identified LsEVs was confirmed using a confocal microscope, and enzyme-linked immunosorbent assay was employed to analyze the levels of inflammatory cytokines in LPS-challenged RAW264.7 cells. The results revealed that the membrane-enclosed LsEVs were spherical, with diameters ranging from 100-250 nm. The LsEVs with diameters of 111-256 nm contained the greatest amount of cargo. In total, 320 proteins (10-38 kD) were identified in the LsEVs and included anti-inflammatory molecules, such as PrtP proteinase, co-chaperones, and elongation factor Tu, as well as some proteins involved in glycolysis/gluconeogenesis, such as fructose-1,6-bisphosphate aldolase. Enrichment analysis showed these proteins to be related to the terms "metabolic pathway," "ribosome," "glycolysis/gluconeogenesis," "carbohydrate metabolism," and "amino acid metabolism." Furthermore, the LsEVs were internalized by host liver cells and can regulate inflammation. These findings confirm that LsEVs contain various functional proteins that play important roles in energy metabolism, signal transduction, and biosynthesis.

Erythrocyte-derived extracellular vesicles from type 2 diabetes patients induce endothelial dysfunction through arginase 1

Abstract Background The mechanisms driving the development of cardiovascular injury in type 2 diabetes (T2D) remain incompletely understood. We recently demonstrated that red blood cells (RBCs) from patients with T2D (T2D-RBCs) act as mediators of endothelial dysfunction, but the mechanisms underlying this interaction are not clarified. We found that RBCs via upregulation of arginase 1 attenuate nitric oxide bioavailability and endothelial function. It is increasingly clear that extracellular vesicles (EVs) are actively secreted by practically all cell types, including RBCs, and represent a novel mechanism of intercellular communication. Purpose This study aimed to determine whether EVs derived from T2D-RBCs are involved as mediators in vascular injury through the signalling of arginase 1. Methods T2D-RBCs and RBCs from age-matched healthy controls (H-RBCs) were isolated and incubated with Krebs-Henseleit buffer (20% haematocrit). Following 18h incubation, the conditioned medium was collected for EV isolation using sequential ultracentrifugation and membrane affinity column. EV concentration was measured by nanoparticle tracking analysis. Aortas isolated from wild-type mice were incubated with EVs derived from T2D-RBCs and H-RBCs for 18h. Endothelium-dependent and -independent relaxations (EDR and EIDR, respectively) of the aortae were evaluated in a wire myograph. The involvement of arginase was investigated by the addition of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) either to the 18h co-incubation of EVs with the aortic segments to selectively investigate the contribution of EV-derived arginase (ABH, 10 mM) or to the aortae following the 18h co-incubation to selectively target vascular arginase (ABH, 100 µM). All animal experiments were performed according to the principles of laboratory animal care (NIH Publication no. 85-23 revised 1985) and human procedures according to the declaration of Helsinki and approved by the Swedish Ethical Review Authority. Results T2D-RBCs had a ten times lower EV concentration compared to H-RBCs. T2D-RBC-derived EVs isolated using both methods significantly impaired EDR (Fig. 1A-C), whereas EIDR was not affected. This effect was observed irrespective of if the same volume or concentration of EVs were administered. Inhibition of arginase with ABH during the co-incubation with EVs derived from T2D-RBCs completely prevented the impairment of EDR induced by the EVs (Fig. 1D). Administration of ABH to the aortae following the co-incubation also attenuated the impairment of EDR. Immunohistochemical staining revealed upregulation of arginase 1 (Fig. 2A-B) but not arginase 2 (Fig. 2C-D) in the vasculature following incubation with EVs from T2D-RBCs. Conclusion EVs derived from T2D-RBCs induce endothelial dysfunction, and arginase 1 derived from these EVs mediates this endothelial dysfunction. These results shed new important light on the mechanism underlying vascular injury mediated by RBCs in T2D.

EVs derived from physiologically loaded myocardial slices affect remodelling of overloaded myocardial slices

Abstract Introduction Mechanical load is an important determinant of cardiac output, and excessive mechanical load can lead to heart remodelling, and ultimately failure. Cardiac intercellular communication is partially mediated by extracellular vesicles (EVs), which are 30-200 nm sized nanoparticles acting as shuttles of many bioactive cargoes that modulate cell behaviour, such as microRNAs. Living myocardial slices are an ideal platform to study how mechanical load affect intercellular communication through EVs, as they can be manipulated to obtain different degrees of preload and are an organotypic multicellular platform that retains the electrophysiology and extracellular matrix of the adult myocardial tissue. Purpose The aim of the study is to investigate how EVs derived from physiologically-loaded slices modulate the myocardial remodelling of overloaded slices. Methods 300 µm slices were obtained from the left ventricle of 300-350 g male Sprague Dawley rats. Mechanical load was applied by stretching the slices to either 2.2 or 2.4 µm of their sarcomere length, to recapitulate physiological and overloaded condition, respectively. Following 48-hours of biomimetic culture, force-stretch relationship of the slices was assessed, the tissue collected for RNA and protein extraction and the media of physiological slices was harvested for EVs isolation by size exclusion chromatography. Physiological EVs were then applied onto overloaded slices (1x108 EVs/slice) and cultured for 48-hours to assess force-stretch relationship, and protein and mRNA expression associated with cardiac remodelling. Physiological and overloaded EVs content was analysed through a microRNA array. Results Physiological slices had a significantly higher contractility (P < 0.05), higher blood vessel density (P < 0.0001) and lower apoptosis (P<0.01) compared to overloaded slices. Application of physiological EVs on overloaded slices restored their contractility (P<0.05), attenuated apoptosis by reducing protein PARP-1 expression (P<0.05) and reduced blood vessels density loss (P< 0.01), as shown by Isolectin B4 staining and increased mRNA levels of VEGF. microRNAs analysis showed significant downregulation of miR-346 and miR-16-5p and upregulation of miR-143-3p miR-378a-3p in physiological EVs, compared to overloaded. The upregulation or downregulation of these microRNAs was positively associated with cardiac apoptosis and migration of vascular smooth muscle cells. Conclusions Physiological EVs affect remodelling in overloaded slices. This is likely due to an effect on apoptosis or to an indirect effect on endothelial cells. Further validation and mechanistic studies with miRNAs selected candidates may reveal interesting therapeutic targets for heart failure.

Abstract 14283: Brain-Mediated Cardioprotective Actions of Leptin: Potential Role of Brown Adipose Tissue and Its Release of Extracellular Vesicles

Background: We recently demonstrated that leptin has powerful central nervous system (CNS) actions that dramatically improve cardiac function following ischemia/reperfusion (IR) injury. How the brain communicates with the heart during CNS leptin infusion to improve cardiac function is unknown. One potential mechanism is activation of brown adipose tissue (BAT) which may be an important source of extracellular vesicles (EVs) carrying cardioprotective microRNAs (miR), including miR-125b-5p. Hypothesis: We investigated whether BAT contributes to the cardioprotective effect of CNS leptin infusion and the potential role of EVs carrying the miR-125b-5p in this protection. Methods: Male Sprague-Dawley rats were submitted to interscapular BAT ablation (iBATx) and instrumented with an intracerebroventricular (ICV) cannula in the brain lateral ventricle. After recovery and baseline assessment of cardiac function by echocardiography (ECHO), myocardial IR was induced by temporary ligation of the left anterior descending coronary artery. Vehicle (saline, 0.5 μL/hr) or leptin (0.62 μg/hr) was infused ICV chronically for 28 days using osmotic pumps. Cardiac function was assessed weekly by ECHO. Another set of animals with intact iBAT was treated with ICV leptin or vehicle for 4 weeks and had their iBAT collected for EVs isolation and quantification. Expression of the miR-125b-5p was measured in the heart of leptin treated rats with intact iBAT or iBATx. Results: Compared to vehicle-treated animals, ICV leptin infusion increased iBAT-derived EVs and removal of iBAT prevented leptin’s CNS-mediated cardioprotective effects after IR injury as indicated by a lack of improvement in ejection fraction (26±6 vs 41±3%) and stroke volume (162±31 vs 275±19μL) at week 4 post-IR. Expression of the mature miR-125b-5p was increased in the heart of ICV leptin treated rats and iBATx abolished this effect. Conclusion: These results indicate that the BAT plays an important role in mediating the cardioprotective effect of CNS leptin administration after IR injury and suggest that iBAT-derived EVs may be involved in delivering the miR-125b-5p to the heart during central leptin infusion. AHA 835218, 1R01HL163076, P20GM104357, and P20GM104320-07

Abstract 17376: Comprehensive Quantitation of Cardiac Tissue Derived Extracellular Vesicles Regulating Adipose Tissue Metabolism

The heart is considered as a recipient of signals from the periphery to fine tune blood pumping in response to physiological and pathological changes. Cardiac secretory function consisting of proteins termed as cardiomyokines has founded the concept of heart mediated organ crosstalk . Studies have shown extracellular vesicles (EVs) are major players regulating organ crosstalk between peripheral organs and the heart. Nevertheless, whether cardiac tissue derived EVs with unique composition deliver messages to peripheral organs remains unknown. Paired serum samples isolated from coronary sinus (CS) and aorta (AO) of dogs under normal and tachypacing induced heart failure were used for EV isolation, characterization and miRNA array analysis. Transplantation of EV from CS and AO samples under normal and heart failure conditions in wild type mice was done to determine whole body metabolism by CLAMs and modulation of adipose tissue metabolism together with in vitro functional assay in cultured adipocytes. Results showed heart failure promotes increased secretion of EVs in the CS compared to AO blood suggesting heart produces more EVs under diseased conditions. miRNAs array analysis identified top elevated miRNAs as miRNAs 16, 92 and 137 significantly enriches in CS EVs under heart failure conditions. Measurement of miRNAs 16, 92, 137 is different tissues showed significantly increased expression in brown adipose tissue (BAT) compared to all other tissues assessed. Next, 3TC-LI adipocytes treated with CS EVs from heart failure dogs showed decreased OXPHOS measured by Seahorse, reduced expression of mitochondrial markers and low BODIPY labelling together with increase in whole body oxygen consumption rates, food intake and energy expenditure as measured by CLAMS. Mechanistically, EV derived miRNAs 16, 92, 137 significant transcriptomic changes BAT assessed by bulk RNA-sequencing. Top altered genes identified in BAT were UQCR10, CPT1a and PGC1a after administration of CS EVs from heart failure animals and are binding targets for miRNAs 16, 92 and 137 respectively. In conclusion, cardiac tissue communicates to adipose tissue to regulate metabolism by releasing EVs derived miRNAs enriched differently in response to heart failure or under normal conditions.

Establishing the Callus-Based Isolation of Extracellular Vesicles from Cissus quadrangularis and Elucidating Their Role in Osteogenic Differentiation.

Extracellular vesicles (EVs) are nano-sized vehicles secreted by all live cells to establish communication with adjacent cells. In recent years, mammalian EVs (MEVs) have been widely investigated for their therapeutic implications in human disease conditions. As the understanding of MEV composition and nature is advancing, scientists are constantly exploring alternatives for EV production with similar therapeutic potential. Plant-derived exosome-like nanovesicles (PDEVs) may be a better substitute for MEVs because of their widespread sources, cost-effectiveness, and ease of access. Cissus quadrangularis (CQ), known as "bone setter or Hadjod", is a perennial plant utilized for its osteogenic potential. Its crude powder extract formulations are widely used as tablets and syrups. The present work elucidates the isolation of exosome-like nanovesicles (henceforth exosomes) from the culture supernatants of an in vitro cultured callus tissue derived from CQ. The physical and biological properties of the exosomes were successfully investigated using different characterization techniques. The therapeutic potential of the CQ exosomes was found to ameliorate the wound scratch injury and oxidative stress conditions in human-derived mesenchymal stem cells (hMSCs) and the pre-osteoblast (MC3T3) cell line. These exosomes also induced the proliferation and differentiation of hMSCs, as observed by alkaline phosphatase activity. These findings may serve as a proof of concept for further investigating the CQ exosomes as a nanocarrier for drug molecules in various therapeutic bone applications.

Does the interplay between human endogenous retrovirus K and extracellular vesicles contribute to aging?

The role of extracellular vesicles (EVs), including retroviral-like particles (RVLPs), in pathogenic processes is currently a subject of active investigation. Several studies have identified mechanistic links between the increased presence of EVs and the process of senescence. A recent study reveals that the reverse transcribed complementary DNA (cDNA) of a human endogenous retroviral sequence can activate the innate immune system and result in tissue damage and/or the spread of cellular senescence to distant tissues. Several studies have linked EVs to age-related diseases, such as Alzheimer's disease and Parkinson's disease, and have included isolation of EVs from individuals with these diseases. Loss of epigenetic regulation, immune activation, and environmental stimuli can all lead to the expression of endogenous retroviruses and the incorporation of their proteins and transcripts into EVs. In addition, EVs disseminating these endogenous retroviral components have now been shown to act in a paracrine manner in multiple human diseases. Further investigation of the connection between EVs containing endogenous retroviral protein products or nucleotides should be pursued in models of age-related diseases.

Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis.

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of cell, they provide a real-time snapshot of host cells and contain a wealth of molecular information, including proteins, in particular those with post-translational modifications (PTMs) such as phosphorylation, as the main player of cellular functions and disease onset and progression. However, the isolation of EVs from biofluids remains challenging due to low yields and impurities from current EV isolation methods, making the downstream analysis of EV cargo, such as EV phosphoproteins, difficult. Here, we describe a rapid and effective EV isolation method based on functionalized magnetic beads for EV isolation from biofluids such as human urine and downstream proteomics and phosphoproteomics analysis following EV isolation. The protocol enabled a high recovery yield of urinary EVs and sensitive profiles of EV proteome and phosphoproteome. Furthermore, the versatility of this protocol and relevant technical considerations are also addressed here.