Electrostatic Surface Properties of Blood and Semen Extracellular Vesicles: Implications of Sialylation and HIV-Induced Changes on EV Internalization.
ABSTRACT: Although extracellular vesicle (EV) surface electrostatic properties (measured as zeta potential, ?-potential) have been reported by many investigators, the biophysical implications of charge and EV origin remains uncertain. Here, we compared the ?-potential of human blood EVs (BEVs) and semen EVs (SEVs) from 26 donors that were HIV-infected (HIV+, n = 13) or HIV uninfected (HIV-, n = 13). We found that, compared to BEVs that bear neutral surface charge, SEVs were significantly more negatively charged, even when BEVs and SEVs were from the same individual. Comparison of BEVs and SEVs from HIV- and HIV+ groups revealed subtle HIV-induced alteration in the ?-potential of EVs, with the effect being more significant in SEVs (??-potential = -8.82 mV, p-value = 0.0062) than BEVs (??-potential = -1.4 mV, p-value = 0.0462). These observations were validated by differences in the isoelectric point (IEP) of EVs, which was in the order of HIV + SEV ? HIV-SEV ? HIV + BEV ? HIV-BEV. Functionally, the rate and efficiency of SEV internalization by the human cervical epithelial cell line, primary peripheral blood lymphocytes, and primary blood-derived monocytes were significantly higher than those of BEVs. Mechanistically, removal of sialic acids from the surface of EVs using neuraminidase treatment significantly decreased SEV's surface charge, concomitant with a substantial reduction in SEV's internalization. The neuraminidase effect was independent of HIV infection and insignificant for BEVs. Finally, these results were corroborated by enrichment of glycoproteins in SEVs versus BEVs. Taken together, these findings uncover fundamental tissue-specific differences in surface electrostatic properties of EVs and highlight the critical role of surface charge in EV/target cell interactions.
Project description:Extracellular vesicles (EVs) are important mediators of intercellular communication. Their role in disease processes, uncovered mostly over the last two decades, makes them potential biomarkers, leading to a need to fundamentally understand EV biology. Direct visualization of EVs can provide insights into EV behavior, but current labeling techniques are often restricted by false-positive signals and rapid photobleaching. Hence, we developed a method of labeling EVs through conjugation with quantum dots (QDs)-high photoluminescent nanosized semi-conductors-using click chemistry. We showed that QD-EV conjugation could be tailored by altering QD to EV ratio or by using a catalyst. This conjugation chemistry was stable in a biological environment and upon storage for up to a week. Using size-exclusion chromatography, QD-EV conjugates could be separated from unconjugated QDs, enabling EV-specific signal detection. We demonstrate that these QD-EV conjugates can be live- and fixed-imaged in high resolution on cells and in tissue sheets, and the conjugates have better photostability compared with the commonly used EV dye DiI. We labeled two distinct EV populations: human semen EVs (sEVs) from fresh semen samples donated by healthy volunteers and brain EVs (bEVs) from excised rat brain tissues. We visualized QD-sEVs in epithelial sheets isolated from human vaginal mucosa and time-lapse imaged QD-bEV interactions with microglial BV-2 cells. The development of the QD-EV conjugate will benefit the study of EV localization, movement, and function and accelerate their potential use as biomarkers, therapeutic agents, or drug-delivery vehicles.
Project description:Blood extracellular vesicles (BEVs) carry bioactive cargo (proteins, genetic materials, lipids, licit, and illicit drugs) that regulate diverse functions in target cells. The cannabinoid drug delta-9-tetrahydrocannabinol (THC) is FDA approved for the treatment of anorexia and weight loss in people living with HIV. However, the effect of THC on BEV characteristics in the setting of HIV/SIV infection needs to be determined. Here, we used the SIV-infected rhesus macaque model of AIDS to evaluate the longitudinal effects of THC (THC/SIV) or vehicle (VEH/SIV) treatment in HIV/SIV infection on the properties of BEVs. While BEV concentrations increased longitudinally (pre-SIV (0), 30, and 150 days post-SIV infection (DPI)) in VEH/SIV macaques, the opposite trend was observed with THC/SIV macaques. SIV infection altered BEV membrane properties and cargo composition late in infection, since i) the electrostatic surface properties (zeta potential, ? potential) showed that RM BEVs carried negative surface charge, but at 150 DPI, SIV infection significantly changed BEV ? potential; ii) BEVs from the VEH/SIV group altered tetraspanin CD9 and CD81 levels compared to the THC/SIV group. Furthermore, VEH/SIV and THC/SIV BEVs mediated divergent changes in monocyte gene expression, morphometrics, signaling, and function. These include altered tetraspanin and integrin ?1 expression; altered levels and distribution of polymerized actin, FAK/pY397 FAK, pERK1/2, cleaved caspase 3, proapoptotic Bid and truncated tBid; and altered adhesion of monocytes to collagen I. These data indicate that HIV/SIV infection and THC treatment result in the release of bioactive BEVs with potential to induce distinct structural adaptations and signaling cues to instruct divergent cellular responses to infection.
Project description:Extracellular vesicles (EVs) mediate targeted cellular interactions in normal and pathophysiological conditions and are increasingly recognised as potential biomarkers, therapeutic agents and drug delivery vehicles. Based on their size and biogenesis, EVs are classified as exosomes, microvesicles and apoptotic bodies. Due to overlapping size ranges and the lack of specific markers, these classes cannot yet be distinguished experimentally. Currently, it is a major challenge in the field to define robust and sensitive technological platforms being suitable to resolve EV heterogeneity, especially for small EVs (sEVs) with diameters below 200 nm, i.e. smaller microvesicles and exosomes. Most conventional flow cytometers are not suitable for the detection of particles being smaller than 300 nm, and the poor availability of defined reference materials hampers the validation of sEV analysis protocols. Following initial reports that imaging flow cytometry (IFCM) can be used for the characterisation of larger EVs, we aimed to investigate its usability for the characterisation of sEVs. This study set out to identify optimal sample preparation and instrument settings that would demonstrate the utility of this technology for the detection of single sEVs. By using CD63eGFP-labelled sEVs as a biological reference material, we were able to define and optimise IFCM acquisition and analysis parameters on an Amnis ImageStreamX MkII instrument for the detection of single sEVs. In addition, using antibody-labelling approaches, we show that IFCM facilitates robust detection of different EV and sEV subpopulations in isolated EVs, as well as unprocessed EV-containing samples. Our results indicate that fluorescently labelled sEVs as biological reference material are highly useful for the optimisation of fluorescence-based methods for sEV analysis. Finally, we propose that IFCM will help to significantly increase our ability to assess EV heterogeneity in a rigorous and reproducible manner, and facilitate the identification of specific subsets of sEVs as useful biomarkers in various diseases.
Project description:Extracellular vesicles (EVs) have raised high expectations as a novel class of diagnostics and therapeutics. However, variabilities in EV isolation methods and the unresolved structural complexity of these biological-nanoparticles (sub-100 nm) necessitate rigorous biophysical characterization of single EVs. Here, using atomic force microscopy (AFM) in conjunction with direct stochastic optical reconstruction microscopy (dSTORM), micro-fluidic resistive pore sizing (MRPS), and multi-angle light scattering (MALS) techniques, we compared the size, structure and unique surface properties of breast cancer cell-derived small EVs (sEV) obtained using four different isolation methods. AFM and dSTORM particle size distributions showed coherent unimodal and bimodal particle size populations isolated via centrifugation and immune-affinity methods respectively. More importantly, AFM imaging revealed striking differences in sEV nanoscale morphology, surface nano-roughness, and relative abundance of non-vesicles among different isolation methods. Precipitation-based isolation method exhibited the highest particle counts, yet nanoscale imaging revealed the additional presence of aggregates and polymeric residues. Together, our findings demonstrate the significance of orthogonal label-free surface characteristics of single sEVs, not discernable via conventional particle sizing and counts alone. Quantifying key nanoscale structural characteristics of sEVs, collectively termed 'EV-nano-metrics' enhances the understanding of the complexity and heterogeneity of sEV isolates, with broad implications for EV-analyte based research and clinical use.
Project description:Serum is an abundant and accessible source of circulating extracellular vesicles (EVs). Serum-EV (sEV) pro-angiogenic capability and mechanisms are herein analyzed using an in vitro assay which predicts sEV angiogenic potential in vivo. Effective sEVs (e-sEVs) also improved vascular remodeling and prevented muscle damage in a mouse model of acute hind limb ischemia. e-sEV angiogenic proteomic and transcriptomic analyses show a positive correlation with matrix-metalloproteinase activation and extracellular matrix organization, cytokine and chemokine signaling pathways, Insulin-like Growth Factor and platelet pathways, and Vascular Endothelial Growth Factor signaling. A discrete gene signature, which highlights differences in e-sEV and ineffective-EV biological activity, was identified using gene ontology (GO) functional analysis. An enrichment of genes associated with the Transforming Growth Factor beta 1 (TGF?1) signaling cascade is associated with e-sEV administration but not with ineffective-EVs. Chromatin immunoprecipitation analysis on the inhibitor of DNA binding I (ID1) promoter region, and the knock-down of small mother against decapentaplegic (SMAD)1-5 proteins confirmed GO functional analyses. This study demonstrates sEV pro-angiogenic activity, validates a simple, sEV pro-angiogenic assay which predicts their biological activity in vivo, and identifies the TGF?1 cascade as a relevant mediator. We propose serum as a readily available source of EVs for therapeutic purposes.
Project description:Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy-based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently-available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non-toxic, and/or clinically-amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non-immunogenicity, biodegradability, and low toxicity. Chitosan-precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic-based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture-conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method.
Project description:BACKGROUND:Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells. METHODS:To probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for ?v?3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments. RESULTS:We find that SEVs secreted from MDA-MB-231 breast cancer cells carry ?v?3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of ?v?3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content. CONCLUSION:In summary, our findings demonstrate for the first time a key role of ?v?3 integrin in cell-cell communication through SEVs. Video Abstract.
Project description:Hypoxic-Ischemic Encephalopathy (HIE) in the brain is the leading cause of morbidity and mortality in neonates and can lead to irreparable tissue damage and cognition. Thus, investigating key mediators of the HI response to identify points of therapeutic intervention has significant clinical potential. Brain repair after HI requires highly coordinated injury responses mediated by cell-derived extracellular vesicles (EVs). Studies show that stem cell-derived EVs attenuate the injury response in ischemic models by releasing neuroprotective, neurogenic, and anti-inflammatory factors. In contrast to 2D cell cultures, we successfully isolated and characterized EVs from whole brain rat tissue (BEV) to study the therapeutic potential of endogenous EVs. We showed that BEVs decrease cytotoxicity in an ex vivo oxygen glucose deprivation (OGD) brain slice model of HI in a dose- and time-dependent manner. The minimum therapeutic dosage was determined to be 25 μg BEVs with a therapeutic application time window of 4-24 h post-injury. At this therapeutic dosage, BEV treatment increased anti-inflammatory cytokine expression. The morphology of microglia was also observed to shift from an amoeboid, inflammatory phenotype to a restorative, anti-inflammatory phenotype between 24-48 h of BEV exposure after OGD injury, indicating a shift in phenotype following BEV treatment. These results demonstrate the use of OWH brain slices to facilitate understanding of BEV activity and therapeutic potential in complex brain pathologies for treating neurological injury in neonates.
Project description:Staphylococcus aureus enhances neutrophil extracellular vesicle (EV) production. To investigate whether S. aureus viability influences EV biogenesis, EVs were isolated from human neutrophils incubated with viable bacteria (bEVs) or heat-killed bacteria (heat-killed EVs). Protein analysis, nanoparticle tracking and transmission electron microscopy showed comparable EV production between subsets, and both viable and nonviable bacteria were also detected in respective EV subsets. As anticipated, S. aureus, as well as bEVs with viable bacteria, were proinflammatory, and killing bacteria with gentamicin reduced cytokine production to baseline levels. Although heat-killed bacteria induced macrophage IL-6 production, heat-killed EVs did not. Additionally, we found that human and bacterial DNA associated with bEVs, but not heat-killed EVs, and that the DNA association could be partially decreased by disrupting electrostatic interactions. We investigated the potential for DNA isolated from EVs (EV-DNA) or EVs to cause inflammation. Although liposomal encapsulation of EV-DNA increased IL-6 production from baseline by 7.5-fold, treatment of bEVs with DNase I had no effect on IL-6 and IL-1β production, suggesting that the DNA did not contribute to the inflammatory response. Filtered EVs, which lacked DNA and associated bacteria, exhibited less proinflammatory activity relative to bEVs, and enhanced macrophage expression of CD86 and HLA-DR. Ultimately, we show that bEVs isolated by differential centrifugation co-purify with bacteria and DNA, and studying their concerted activity and relative contribution to immune response is important to the study of host-pathogen interactions.
Project description:Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues.