Characterization and function of medium and large extracellular vesicles from plasma and urine by surface antigens and Annexin V

Antibodies and other reagents

The following monoclonal antibodies against human surface antigens were used in this study: anti-CD5(clone: L17F12), anti-CD15 (clone: W6D3), anti-CD41(clone: HIP8), anti-CD45(clone: HI30), anti-CD59 (clone: p282), anti-CD61 (clone: VI-PL2), anti-CD105 (clone: 43A3), anti-CD146(clone: P1H12), anti-CD235a (clone: HI264), anti-CD10 (clone: HI10a), anti-CD13 (clone: WM15), anti-CD26 (clone: BA5b), anti-CD227 (MUC1) (clone: 16A). All antibodies were purchased from BioLegend^® (San Diego, CA). FITC-conjugated Annexin V was purchased from BD Biosciences (New Jersey, USA). We used the SPHERO™ Nano Fluorescent Particle Size Standard Kit, Yellow (diameters 0.22, 0.45, 0.88 and 1.35 µm) from Spherotech Inc. for size validation. Normal mouse IgG was purchased from Wako Chemicals (Tokyo, Japan). APC-conjugated normal mouse IgG was produced using the Mix-n-Stain™ APC Antibody Labeling kit from Biotium Inc. Dithiothreitol (DTT) was purchased from Wako Chemicals (Tokyo, Japan). We conducted phase transfer surfactant experiments using “MPEX PTS Reagents for MS” purchased from GL Sciences Inc. and “Trypsin, TPCK Treated” purchased from Thermo Fisher Scientific™. Iodoacetamide was purchased from Wako Chemicals (Tokyo, Japan).

Samples

All studies were approved by the Institutional Review Board of the Kyushu University Hospital, Kyushu University (29-340). Blood samples were collected from 20 male and female participants (23–48 years of age) who were apparently healthy. We received the informed consent from all participants of this study. Samples were collected using a 22-gauge butterfly needle and a slow-fill syringe. After discarding the initial 2–3 mL, blood was dispensed into collection tubes containing ethylenediamine tetra acetic acid (EDTA) (1.6 mg/mL blood). Urine was collected from 20 male healthy subjects (23–46 years of age). The first morning void urine was used for the experiments. The urine samples were collected in a sterile container. In particular, we confirmed that sample used for analysis by flow cytometer were derived from healthy donors by measuring blood count and creatinine in blood and total urine protein (Table S1).

Isolation of plasma m/lEVs

Essentially platelet-free plasma (PFP) was prepared from EDTA-treated blood by double centrifugation at 2, 330 ×g for 10 min. To assess residual platelets remaining in this sample, we measured platelet number using the ADVIA^® 2120i Hematology System (SIEMENS Healthineers, Erlangen Germany). The number of platelets in this sample was below the limit of detection (1 ×10³ cells/µL). We used a centrifugation method to obtain m/lEVs. In an effort to ensure our approach could be applied to clinical testing, we chose a simple and easy method for pretreatment. In an ISEV position paper (Mateescu et al., 2017), Thery’s group referred to vesicles sedimenting at 10 0, 000 ×g as “small EVs” rather than exosomes, those pelleting at intermediate speed (lower than 2 0, 000 ×g) as “medium EVs” (including microvesicles and ectosomes) and those pelleting at low speed (e.g.,  2000 ×g) as “large EVs”. Because these definitions are less biologically meaningful but more experimentally tractable than previously-applied exosome/microvesicle definitions, we attempted biological characterization through subsequent shotgun and flow cytometry analysis.

In flow cytometric analysis, the volume of PFP used in each assay was 0.6 mL from each donor. In electron microscopy, the volume of PFP used was three mL. Samples were independent and were treated individually prior to each measurement. PFP was centrifuged at 18, 900 ×g for 30 min in a fixed-angle rotor. The m/lEV pellet obtained after centrifugation was reconstituted by vortex mixing (1–2 min) with an equivalent volume of Dulbecco’s phosphate-buffered saline (DPBS), pH 7.4. The solution was centrifuged at 18, 900 ×g for 30 min again and the supernatant was discarded.

Isolation of urinary m/lEVs

For isolation of urinary m/lEVs, we modified a urinary exosome extraction protocol (Fernandez-Llama et al., 2010). The centrifugation conditions were identical for plasma and urine so that the size and the density of m/lEVs were similar, enabling comparison of plasma and urinary m/lEVs.

In flow cytometric analysis, the volume of urine used for each assay was 1.2 mL from each donor. In electron microscopy, the volume of urine used was 15 mL. Samples were independent and were treated individually prior to each measurement. Collected urine was centrifuged at 2, 330 ×g for 10 min twice. The supernatant was centrifuged at 18, 900 ×g for 30 min in a fixed-angle rotor. The m/lEV pellet obtained from centrifugation was reconstituted by vortex mixing (1–2 min) with 0.2 mL of DPBS followed by incubation with DTT (final concentration 10 mg/mL) at 37 °C for 10–15 min. The samples were centrifuged again at 18, 900 ×g for 30 min and the supernatant was discarded. Addition of DTT, a reducing agent, reduced the formation of Tamm-Horsfall protein (THP) polymers. THP monomers were removed from m/lEVs after centrifugation. DTT-containing DPBS solutions were filtered through 0.1-µm filters (Millipore).

Flow cytometric analysis of m/lEVs

After resuspending m/lEV pellets in 60 µL of DPBS, we added saturating concentrations of several labelled antibodies, Annexin V and normal mouse IgG and incubated the tubes in the dark, without stirring, for 15–30 min at room temperature. In one case, we added labelled antibodies directly to 60 µL of PFP for staining. We resuspended stained fractions in Annexin V binding buffer (BD Biosciences: 10 mM HEPES, 0.14 mM NaCl, 2.5 mM CaCl₂, pH 7.4) for analysis by flow cytometry. DPBS and Annexin V binding buffer were filtered through 0.1-µm filters (Millipore). Flow cytometry was performed using a FACSVerse™ flow cytometer (BD Biosciences). The flow cytometer was equipped with 405 nm, 488 nm and 638 nm lasers to detect up to 13 fluorescent parameters. The flow rate was 12 µL /min. Forward scatter voltage was set to 381, side scatter voltage was set to 340, and each threshold was set to 200. Details of excitation (Ex.) and emission (Em.) wavelengths as well as voltages described in supplements Fig. Flow cytometry was performed using FACSuite™ software (BD Biosciences) and data were analyzed using FlowJo software. The authors have applied for the following patents for the characterization method of m/lEVs isolated from plasma and urine with a flow cytometer: JP2018-109402(plasma) and JP2018-109403(urine).

Nanoparticle tracking analysis (NTA)

NTA measurements were performed using a NanoSight LM10 (NanoSight, Amesbury, United Kingdom). After resuspending mEV pellets in 50 µL of DPBS, samples were diluted eight-fold (plasma) and 100-fold (urinary) with PBS prior to measurement. Particles in the laser beam undergo Brownian motion and videos of these particle movements are recorded. NTA 2.3 software then analyses the video and determines the particle concentration and the size distribution of the particles. Twenty-five frames per second were recorded for each sample at appropriate dilutions with a “frames processed” setting of 1,500. The detection threshold was set at “7 Multi” and at least 1,000 tracks were analyzed for each video.

Electron microscopy

For immobilization, we added 100 µL of PBS and another 100 µL of immobilization solution (4% paraformaldehyde, 4% glutaraldehyde, 0.1 M phosphate buffer, pH 7.4) to m/lEV pellets. After stirring, we incubated at 4 °C for 1 h. For negative staining, the samples were adsorbed to formvar film-coated copper grids (400 mesh) and stained with 2% phosphotungstic acid, pH 7.0, for 30 s. For observation and imaging, the grids were observed using a transmission electron microscope (JEM-1400Plus; JEOL Ltd., Tokyo, Japan) at an acceleration voltage of 100 kV. Digital images (3,296 × 2,472 pixels) were taken with a CCD camera (EM-14830RUBY2; JEOL Ltd., Tokyo, Japan).

Protein digestion

We used approximately 50 mL of pooled healthy plasma and 100 mL of pooled healthy male urine from five healthy subjects for digestion of m/lEVs.

In plasma the following process is the same as “Isolation of plasma m/lEVs” section. We repeated 18, 900 ×g centrifugation washing steps three times to reduce levels of contaminating free plasma proteins and small EVs for shotgun analysis. After the last centrifugation, we removed supernatants and froze the samples.

In urine the following process is the same as “Isolation of urinary m/lEVs” section. We repeated washing steps twice (after DTT treatment) to reduce levels of contaminating free urinary proteins and small EVs for shotgun analysis. We removed supernatants and froze the samples.

To discover characterizing surface antigen by flowcytometry, the sample was digested using a phase transfer surfactant-aided procedure so that many hydrophobic membrane proteins could be detected (Chen et al., 2017). The precipitated frozen fractions of plasma and urine were thawed at 37 °C, and then m/lEVs were solubilized in 250 µL of lysis buffer containing 12 mM sodium deoxycholate and 12 mM sodium lauroyl sarcosinate in 100 mM Tris-HCl, pH 8.5. After incubating for 5 min at 95 °C, the solution was sonicated using an ultrasonic homogenizer. Protein concentrations of the solutions were measured using a bicinchoninic acid assay (Pierce™ BCA Protein Assay Kit; Thermo Fisher Scientific).

Twenty microliters of the dissolved pellet (30 µg protein) were used for protein digestion. Proteins were reduced and alkylated with 1 mM DTT and 5.5 mM iodoacetamide at 25 °C for 60 min. Trypsin was added to a final enzyme:protein ratio of 1:100 (wt/wt) for overnight digestion. Digested peptides were acidified with 0.5% trifluoroacetic acid (final concentration) and 100 µL of ethyl acetate was added for each 100 µL of digested m/lEVs. The mixture was shaken for 2 min and then centrifuged at 1 5, 600 ×g for 2 min to obtain aqueous and organic phases. The aqueous phase was collected and desalted using a GL-Tip SDB column (GL Sciences Inc).

LC-MS/MS analysis

Digested peptides were dissolved in 40 µL of 0.1% formic acid containing 2% (v/v) acetonitrile and 2 µL were injected into an Easy-nLC 1000 system (Thermo Fisher Scientific). Peptides were separated on an Acclaim PepMap™ RSLC column (15 cm ×50 µm inner diameter) containing C18 resin (2 µm, 100 Å; Thermo Fisher Scientific™), and an Acclaim PepMap™ 100 trap column (two cm ×75 µm inner diameter) containing C18 resin (3 µm, 100 Å; Thermo Fisher Scientific™). The mobile phase consisted of 0.1% formic acid in ultrapure water (buffer A). The elution buffer was 0.1% formic acid in acetonitrile (buffer B); a linear 200 min gradient from 0%–40% buffer B was used at a flow rate of 200 nL/min. The Easy-nLC 1000 was coupled via a nanospray Flex ion source (Thermo Fisher Scientific™) to a Q Exactive™ Orbitrap (Thermo Fisher Scientific™). The mass spectrometer was operated in data-dependent mode, in which a full-scan MS (from 350 to 1,400 m/z with a resolution of 70,000, automatic gain control (AGC) 3E+06, maximum injection time 50 ms) was followed by MS/MS on the 20 most intense ions (AGC 1E+05, maximum injection time 100 ms, 4.0 m/z isolation window, fixed first mass 100 m/z, normalized collision energy 32 eV).

Proteome data analysis

Raw MS files were analyzed using Proteome Discoverer software version 1.4 (Thermo Fisher Scientific™) and peptide lists were searched against the Uniprot Proteomes-Homo sapiens FASTA (Last modified November 17, 2018) using the Sequest HT algorithm. Initial precursor mass tolerance was set at 10 ppm and fragment mass tolerance was set at 0.6 Da. Search criteria included static carbamidomethylation of cysteine (+57.0214 Da), dynamic oxidation of methionine (+15.995 Da) and dynamic acetylation (+43.006 Da) of lysine and arginine residues.

Gene ontology analysis and gene enrichment analysis

We conducted GO analysis using DAVID (https://david.ncifcrf.gov) to categorize the proteins identified by shotgun analysis and used Metascape (http://metascape.org/gp/index.html#/main/step1) for gene enrichment analysis. We uploaded the UNIPROT_ACCESSION No. for each protein.

Extracellular vesicle preparation from isolated erythrocytes

Whole blood was collected by the same method as above and centrifuged at 2, 330 ×g for 10 min. After removal of the buffy coat and supernatant plasma, the remaining erythrocytes were washed three times by centrifugation at 2, 330 ×g for 10 min and the erythrocyte pellet was resuspended in DPBS. EVs were generated from the washed erythrocytes by stimulation in the presence of 2.5 mM CaCl₂ (10 mM HEPES, 0.14 mM NaCl, 2.5 mM CaCl₂, pH 7.4) for 1 h at room temperature under rotating conditions. Erythrocytes were removed by centrifugation at 2, 330 ×g for 10 min and the EV rich supernatant was subsequently centrifuged (18, 900 ×g for 30 min) to pellet the EVs. EVs were resuspended in DPBS.

Dipeptidyl peptidase IV (DPP4:CD26) activity assay

DPP4 activity was measured in the plasma and urine of six individuals (different from plasma donors). The method was previously published in part (Kawaguchi et al., 2010). DPP4 activity was measured via the fluorescence intensity of 7-amino-4-methylcoumarin (AMC) after its dissociation from the synthetic substrate (Gly-Pro-AMC •HBr) catalyzed by DPP4. Experiments were performed in 96-well black plates. Titrated AMC was added to each well to prepare a standard curve. Fluorescence intensity was measured after incubating substrate with urine samples for 10 min. The enzyme reaction was terminated by addition of acetic acid. The fluorescence intensity (Ex. = 380 nm and Em. = 460 nm) was measured using Varioskan Flash (Thermo Fisher Scientific™). DPP4 activity assays were performed by Kyushu Pro Search LLP (Fukuoka, Japan).

Isolation and characterization of m/lEVs from plasma and urine

The workflow for the isolation and enrichment of m/lEVs for flow cytometric analyses is illustrated in Figs. 1A and 1B. m/lEVs from human plasma samples were isolated by high-speed centrifugation, an approach used in previous studies (Jayachandran et al., 2012). For isolation of m/lEVs from urine, DTT, a reducing agent, was used to remove THP polymers because these non-specifically interact with IgGs.

Transmission electron microscopy revealed that almost all m/lEVs were small, closed vesicles with a size of approximately 200 nm that were surrounded by lipid bilayer (Figs. 1C–1H). In plasma, we observed EVs whose membranes were not stained either inside or on the surface (Figs. 1C, 1D); we also observed EVs whose forms were slightly distorted (Fig. 1E). In urine, a group of EVs with uneven morphology and EVs with interior structures were observed (Fig. 1H). Apoptotic bodies, cellular debris, and protein aggregates were not detected.

No EVs with diameters greater than 800 nm were observed by NTA (Fig. S1) and flow cytometry can detect only EVs with diameters larger than 200 nm. Together, these data suggested that we characterized m/lEVs between 200 nm and 800 nm in diameter from plasma and urine by flow cytometry analysis. We observed the m/lEVs less than 100 nm by NTA because of some contamination or degradation after purification (Fig. S1).

Side-scatter events from size calibration beads of (diameters: 0.22, 0.45, 0.88 and 1.35 µm) were resolved from instrument noise using a FACS Verse flow cytometer (Fig. S2A). Inspection of the side-scatter plot indicated that 0.22 µm was the lower limit for bead detection. More than 90% of m/lEVs isolated from plasma and urine showed side-scatter intensities lower than those of 0.88-µm calibration beads (Figs. 2A–2D). m/lEVs were heterogeneous in size, with diameters ranging from 200–800 nm in plasma and urine (Figs. 2A–2D). Fluorescently-labeled mouse IgG was used to exclude nonspecific IgG-binding fractions (Figs. S2B and S2C). In this experiment, we characterized m/lEVs with diameters ranging from 200–800 nm. NTA analysis shows less than 100 nm size particles in the plasma fraction extracted by centrifugation, but we focused on particles over 200 nm using a flow cytometer. Using these methods, we observed an average of 8 ×10⁵ and 1 ×10⁵m/lEVs in each mL of plasma and urine by flow cytometry observation.

Shotgun proteomic analysis of plasma and urine EVs

To analyze the protein components and discover characterizing surface antigen of m/lEVs present in plasma and urine of five healthy individuals, we performed LC-MS/MS proteomic analysis. In this analysis, in order to prevent small EVs contamination, the washing process by centrifugation was increased compared to other analyses (Materials and Methods). A total of 593 and 1,793 proteins were identified in m/lEVs from plasma and urine, respectively (Fig. 3A and Tables S2 and S3). Scoring counts using the SequestHT algorithm for the top 20 most abundant proteins are shown in Tables 1 and 2. We detected cytoskeleton-related protein such as actin, ficolin-3 and filamin and cell–surface antigen such as CD5, band3 and CD41 in plasma. We also identified actin filament-related proteins such as ezrin, radixin, ankylin and moesin which play key roles in cell surface adhesion, migration and organization in both plasma and urine. In urine, several types of peptidases (membrane alanine aminopeptidase or CD13; neprilysin or CD10; DPP4 or CD26) and MUC1 (mucin 1 or CD227) were detected in high abundance, and these proteins were used to characterize m/lEVs by flow cytometric analysis (Table 2 and Table S3). We demonstrated that the isolated m/lEVs showed high expression of tubulin and actinin, while the tetraspanins CD9 and CD81 that are often used as exosome markers were only weakly identified. Especially in plasma, small EV (exosome) markers TSG101, VPS4 and Alix were not observed in this m/lEVs fraction (Table S4). These data suggest that m/lEVs differ from small EVs including exosomes.

As shown in Fig. 3A and Fig. S3, about 10% of urinary EVs proteins were also identified in plasma EVs. Urinary EVs in the presence of blood contaminants were also observed in previous studies (Smalley et al., 2008). These result suggest that m/lEVs in plasma were excreted in the urine via renal filtration and not reabsorbed. Gene ontology analysis of the identified proteins indicated overall similar cellular components in plasma and urine m/lEVs (Fig. 3B). The results of gene set enrichment analysis by metascape are shown for plasma and urine m/lEVs (Figs. 3C, 3D and Tables S5 and S6). The most commonly-observed functions in both plasma and urine were “regulated exocytosis”, “hemostasis” and “vesicle-mediated transport”. In plasma, several functions of blood cells were observed, including “complement and coagulation cascades” and “immune response”. Moreover, analysis of urinary EVs showed several characteristic functions including “transport of small molecules”, ‘metabolic process” and “cell projection assembly”. This may reflect the nature of the kidney, the urinary system and tubular villi. These data demonstrate the power of data-driven biological analyses.

Characterization of plasma EVs by flow cytometry

Next, we characterized m/lEVs in plasma by flow cytometry using antibodies against several surface antigens and Annexin V. To eliminate nonspecific adsorption, we excluded the mouse IgG-positive fraction. (Figs. S2B). Eliminating non-specific reactions to antibodies is important in using human body fluids as diagnostic materials for immunological measurements. By adding mouse IgG-APC to the system, we observed accurate flow cytometry image in which specific surface antigens were recognized by following two points: (1) blocking of non-specific reaction sites, (2) gate-out of positive non-specific reaction. We characterized positive m/lEVs using surface antigens detected by shotgun proteomic analysis and Annexin V (Figs. 4A–4L).

To characterize m/lEVs derived from erythrocytes, T and B cells, macrophages/monocytes, granulocytes, platelets and endothelial cells, we selected nine antigens described in Fig. 4A. Two or more antigens CD235a double-positive and CD45-negative m/lEVs were classified as erythrocyte-derived m/lEVs (Fig. S4B). We confirmed that m/lEVs isolated from erythrocytes in vitro and erythrocytes derived m/lEVs from plasma are characterized by CD59 and CD235a double-positive and CD45-negative (Figs. S5). Determined positive area by addition of EDTA (Figs. S2D), we also show Annexin V staining for the m/lEVs corresponding to these five classifications (Figs. 4B–4L). We integrated these characterizations and assessed the distribution of EV classifications among ten healthy subjects (Fig. 4M). The results suggested that no major differences in the ratios of fractions in these ten subjects and thus these definitions may be used for pathological analysis.

We found that 10% and 35% of m/lEVs were derived from erythrocytes and platelets, respectively. However, only 0.5%, 0.6% and 0.1% of m/lEVs were derived from macrophages, leukocytes and endothelial cells, respectively suggesting that the ratio of m/lEVs of different cellular origins is dependent on the number of cells present in plasma (Fig. 4M). We also observed that most m/lEVs derived from erythrocytes and macrophages were Annexin V positive (Figs. 4N and 4O). By contrast, many Annexin V negative m/lEVs were identified among platelet- and T and B cell-derived m/lEVs (Figs. 4P and 4Q). Especially about erythrocyte-derived m/lEVs other studies have shown high percentages of phosphatidylserine-positive(:Annexin V positive) m/lEVs after red blood cell storage under blood bank conditions that these results are consistent (Gao et al., 2013; Xiong et al., 2011).

In general, it is known that microparicle in blood are known to be exposed to PS on the surface, which is verified by being positive by Annexin5 staining. We found that the degree of exposure of phosphatidylserine to the membrane surface was vary depending on the cell derived from annexin V staining. Thus, the characteristics of m/lEVs can be determined in detail by using AnnexinV and antigenicity. These results suggested that the degree of exposing PS are cell-type specific and that release mechanisms may differ among cell types.

Characterization of urinary EVs by flow cytometry and enzyme activity assay

In urine, we first removed aggregated m/lEVs and residual THP polymers using labelled normal mouse IgG (Figs. S2C). By removing the THP polymer by DTT treatment, many immunological non-specific reactions in flow cytometry observation were eliminated, and the remaining non-specific reactions were completely excluded from the observed image by mouse IgG-positive gating-out (Figs. S2D). To characterize urinary m/lEVs, we used surface antigens detected by shotgun proteomic analysis including CD10 (neprilysin), CD13 (alanine aminopeptidase), CD26 (DPP4) and CD227 (MUC1) (Figs. 5A–5F). Many m/lEVs in the observation area were triple-positive for CD10, CD13 and CD26, but negative for Annexin V (Figs. 5B–5D, Figs. S6). Furthermore, MUC1-positive EVs were both Annexin V positive and negative in roughly equivalent frequencies (Figs. 5B, 5E and 5F). These results suggested that m/lEVs containing peptidases were released by outward budding directly from the cilial membrane of renal proximal tubule epithelial cells. The results of integrating these characterizations and the distribution of EV classifications among ten healthy subjects are shown (Figs. 5G–5I). These data indicated no major differences in the ratio among these populations, suggesting that our methodology was reliable for m/lEV analysis.

We next verified the CD26 peptidase enzyme activities of m/lEVs in plasma and urine from six individuals. We prepared three fractions: (i) “whole,” in which debris were removed after low speed centrifugation, (ii) “m/lEVs” and (iii) “free (supernatant)” both of which were obtained via high speed centrifugation (18, 900 ×g for 30 min) (Fig. 5J). We found that more than 20% of DPP4 activity in whole urine was contributed by the EV fraction (Fig. 5K and Figs. S7). By contrast, there was no peptidase activity associated with plasma m/lEVs (Fig. 5L). These results suggested that functional CD26 peptidase activity is present in m/lEVs in urine, which may be useful for pathological analysis.