Effect of the DASH diet on the sodium-chloride cotransporter and aquaporin-2 in urinary extracellular vesicles.

The dietary approach to stop hypertension (DASH) diet combines the antihypertensive effect of a low sodium and high potassium diet. In particular, the potassium component of the diet acts as a switch in the distal convoluted tubule to reduce sodium reabsorption, similar to a diuretic but without the side effects. Previous trials to understand the mechanism of the DASH diet were based on animal models and did not characterize changes in human ion channel protein abundance. More recently, protein cargo of urinary extracellular vesicles (uEVs) has been shown to mirror tissue content and physiological changes within the kidney. We designed an inpatient open label nutritional study transitioning hypertensive volunteers from an American style diet to DASH diet to examine physiological changes in adults with stage 1 hypertension otherwise untreated (Sacks FM, Svetkey LP, Vollmer WM, Appel LJ, Bray GA, Harsha D, Obarzanek E, Conlin PR, Miller ER 3rd, Simons-Morton DG, Karanja N, Lin PH; DASH-Sodium Collaborative Research Group. N Engl J Med 344: 3-10, 2001). Urine samples from this study were used for proteomic characterization of a large range of pure uEVs (small to large) to reveal kidney epithelium changes in response to the DASH diet. These samples were collected from nine volunteers at three time points, and mass spectrometry identified 1,800 proteins from all 27 samples. We demonstrated an increase in total SLC12A3 [sodium-chloride cotransporter (NCC)] abundance and a decrease in aquaporin-2 (AQP2) in uEVs with this mass spectrometry analysis, immunoblotting revealed a significant increase in the proportion of activated (phosphorylated) NCC to total NCC and a decrease in AQP2 from day 5 to day 11. This data demonstrates that the human kidney's response to nutritional interventions may be captured noninvasively by uEV protein abundance changes. Future studies need to confirm these findings in a larger cohort and focus on which factor drove the changes in NCC and AQP2, to which degree NCC and AQP2 contributed to the antihypertensive effect and address if some uEVs function also as a waste pathway for functionally inactive proteins rather than mirroring protein changes.NEW & NOTEWORTHY Numerous studies link DASH diet to lower blood pressure, but its mechanism is unclear. Urinary extracellular vesicles (uEVs) offer noninvasive insights, potentially replacing tissue sampling. Transitioning to DASH diet alters kidney transporters in our stage 1 hypertension cohort: AQP2 decreases, NCC increases in uEVs. This aligns with increased urine volume, reduced sodium reabsorption, and blood pressure decline. Our data highlight uEV protein changes as diet markers, suggesting some uEVs may function as waste pathways. We analyzed larger EVs alongside small EVs, and NCC in immunoblots across its molecular weight range.

Acute exercise decreases insulin-stimulated extracellular vesicles in conjunction with augmentation index in adults with obesity.

Extracellular vesicles (EVs) are often elevated in obesity and may modulate disease risk. Although acute exercise reduces fasting EVs in adults with obesity, no data exist on insulin-mediated EV responses. This study evaluated the effects of exercise on EV responses to insulin in relation to vascular function. Ten (5M/5F) sedentary adults with obesity (34.3 ± 3.7 kg/m2 ) completed an evening control and acute exercise condition (70% V ̇ O 2 max ${\dot{V}_{{{\rm{O}}_{\rm{2}}}{\rm{max}}}}$ to expend 400 kcal). Following an overnight fast, participants underwent a 2 h euglycaemic-hyperinsulinaemic clamp (90 mg/dl; 40 mU/m2 /min) to determine metabolic insulin sensitivity (M-value), phenotypes of medium- to large-sized EVs, and aortic waveform measures. Endothelial (CD105+ , CD41- /CD31+ )-, leukocyte (CD45+ )-, platelet (CD41+ , CD41+ /31+ )- and tetraspanin (TX+ )-derived EVs, as well as platelet endothelial cell adhesion molecule (CD31+ ), were determined before and after the clamp using high resolution spectral flow cytometry. Although exercise did not alter fasting haemodynamics, it lowered the augmentation index (AIx75, P = 0.024) and increased the M-value (P = 0.042). Further, exercise decreased all fasting EVs (P < 0.01) and decreased insulin-stimulated TX+ (P = 0.060), CD31+ (P = 0.060) and CD41- /31+ (P = 0.045) compared to rest. Interestingly, greater insulin-stimulated decreases in CD41- /31+ were associated with reduced AIx75 during the clamp (r = 0.62, P = 0.059), while insulin-stimulated decreases in CD41+ (r = -0.68, P = 0.031), CD41+ /31+ (r = -0.69, P = 0.262), TX+ (r = -0.66, P = 0.037) and CD31+ (r = -0.69, P = 0.028) correlated with M-value following exercise. Thus, acute exercise may decrease fasting and insulin-stimulated medium- to large-size EVs in conjunction with improved M-value and AIx75. More research is needed to understand effects of exercise on EVs in the regulation of glucose homeostasis and vascular function. KEY POINTS: Extracellular vesicles (EVs) are increased in states of obesity and may play a role in altered insulin sensitivity and blood pressure; aerobic exercise decreases fasting EV concentrations the following day in adults with obesity. This study directly tested the effects of insulin on EVs and how a single bout of exercise impacts these responses. Together, these data highlight the positive effects of a single bout of exercise on fasting and insulin-stimulated EVs, with the latter relating to increased insulin sensitivity and decreased augmentation index. These results support future research identifying EVs as mechanistic factors in glucose regulation and vascular function as well as clinical use of exercise to reduce cardiovascular disease risk.

Insulin infusion decreases medium-sized extracellular vesicles in adults with metabolic syndrome.

Elevated extracellular vesicles (EVs) are associated with glucose dysmetabolism. However, the effects of insulin on EVs and subsequent relationships with insulin sensitivity, substrate oxidation, and inflammation are unknown. We tested the hypothesis that insulin would lower EVs and relate to insulin action. Fifty-one sedentary adults (54.8 ± 1.0 yr; V̇o2peak : 22.1 ± 0.6 mL/kg/min) with metabolic syndrome (MetS) and obesity (36.4 ± 0.65 kg/m2) underwent a 2-h euglycemic-hyperinsulinemic clamp (5 mmol/L; 40 mU/m2/min). Count and size (medium: 200-624 nm; larger: 625-1,000 nm) for total particle count, endothelial- (CD105+), leukocyte- (CD45+), platelet- (CD41+), and tetraspanin- (TX+: CD9/CD81/CD63), as well as platelet endothelial cell adhesion molecule- (CD31+) derived EVs were determined before and following the clamp using Full Spectrum Profiling (FSPM). Size and MESF (molecules of equivalent soluble fluorochrome) data were generated using FCMPASS Software. Fat and carbohydrate oxidation, in addition to high-sensitivity c-reactive protein (hsCRP), were measured to understand insulin effects and associations between EVs, metabolic flexibility, and inflammation. Despite low metabolic insulin sensitivity (M-Value = 2.56 ± 0.17 mg/kg/min), insulin increased carbohydrate (P = 0.015) and decreased fat oxidation (P = 0.048) and hsCRP (P = 0.016) compared with fasting. Insulin also decreased total particle count (P < 0.001), attributable to decreased medium-sized CD105+ (P = 0.052) and CD45+ EVs (P < 0.001). Elevated fasting insulin was associated with reduced insulin-stimulated changes in all EVs phenotypes (P < 0.001). Interestingly, fasting EVs were associated with increased fasting carbohydrate oxidation (all P < 0.05). These findings suggest that insulin decreases medium-sized EVs in conjunction with metabolic flexibility under euglycemic conditions in adults with MetS. More research is needed to determine how therapies alter EV phenotype/size and consequent cardiometabolic risk.NEW & NOTEWORTHY This study is one of the first to investigate the effects of insulin on medium and larger extracellular vesicles (EVs) in relation to metabolic insulin sensitivity and fuel use in adults with metabolic syndrome. Our data suggest that insulin infusion decreases the concentration of total particle counts, mainly due to reductions in medium-sized EVs. Furthermore, EVs, predominantly medium-sized, are inversely associated with metabolic flexibility.

T cell-derived extracellular vesicles are elevated in essential HTN.

Extracellular vesicles (EVs) are novel mediators of cell-to-cell communication and appear to mediate the pathogenesis of hypertension (HTN). However, the mechanisms underlying the involvement of EVs in HTN remain unclear. The adaptive and innate immune systems play an important role affecting the kidney and vasculature in animal models of HTN. Evolving evidence shows that immune cell-derived EVs can modulate the immune system in a paracrine fashion and therefore may mediate the effects of inflammation in the pathogenesis of HTN. Therefore, we aimed to understand if specific subtypes of leukocyte/immune cell-derived EVs are altered in essential HTN using an in vivo model of angiotensin II (ANG II)-induced HTN. After 4 wk of ANG II treatment, EVs were isolated from the blood and kidney. EV origin and counts were characterized with Imaging Flow Cytometry, antibody panels targeting platelets, endothelial cells, and leukocytes including B and T cells, monocytes, and neutrophils. Leukocyte-derived EVs (CD45+) were elevated in the circulation and kidney tissue in ANG II-induced HTN. Subgroup analysis depicted T cell-derived EVs (CD3+) to be significantly elevated in ANG II-induced HTN (3.50e+5 particles/mL) compared with control groups (9.16e+4 particles/mL, P = 0.0106). T cell-derived EVs also significantly correlated with systolic blood pressure levels (r2 = 0.898, P = 0.0012). In summary, leukocyte-derived EVs, and more specifically T cell-derived EVs (CD3+), are elevated in ANG II-induced HTN in the circulation and kidney tissue and correlate well with blood pressure severity. EVs from the circulation and kidney may be sensitive biomarkers for HTN and end-organ damage and may lead to new mechanistic insights in this silent disease.

Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning.

Urinary extracellular vesicles (uEVs) have become a promising source for biomarkers accurately reflecting biochemical changes in kidney and urogenital diseases. Characteristically, uEVs are rich in membrane proteins associated with several cellular functions like adhesion, transport, and signaling. Hence, membrane proteins of uEVs should represent an exciting protein class with unique biological properties. In this study, we utilized uEVs to optimize the Triton X-114 detergent partitioning protocol targeted for membrane proteins and proceeded to their subsequent characterization while eliminating effects of Tamm-Horsfall protein, the most abundant interfering protein in urine. This is the first report aiming to enrich and characterize the integral transmembrane proteins present in human urinary vesicles. First, uEVs were enriched using a "hydrostatic filtration dialysis'' appliance, and then the enriched uEVs and lysates were verified by transmission electron microscopy. After using Triton X-114 phase partitioning, we generated an insoluble pellet fraction and aqueous phase (AP) and detergent phase (DP) fractions and analyzed them with LC-MS/MS. Both in- and off-gel protein digestion methods were used to reveal an increased number of membrane proteins of uEVs. After comparing with the identified proteins without phase separation as in our earlier publication, 199 different proteins were detected in DP. Prediction of transmembrane domains (TMDs) from these protein fractions showed that DP had more TMDs than other groups. The analyses of hydrophobicity revealed that the GRAVY score of DP was much higher than those of the other fractions. Furthermore, the analysis of proteins with lipid anchor revealed that DP proteins had more lipid anchors than other fractions. Additionally, KEGG pathway analysis showed that the DP proteins detected participate in endocytosis and signaling, which is consistent with the expected biological functions of membrane proteins. Finally, results of Western blotting confirmed that the membrane protein bands are found in the DP fraction instead of AP. In conclusion, our study validates the use of Triton X-114 phase partitioning protocol on uEVs for a targeted isolation of membrane proteins and to reduce sample complexity. This method successfully facilitates detection of potential biomarkers and druggable targets in uEVs.

N-linked (N-) glycoproteomics of urinary exosomes. [Corrected].

Epithelial cells lining the urinary tract secrete urinary exosomes (40-100 nm) that can be targeted to specific cells modulating their functionality. One potential targeting mechanism is adhesion between vesicle surface glycoproteins and target cells. This makes the glycopeptide analysis of exosomes important. Exosomes reflect the physiological state of the parent cells; therefore, they are a good source of biomarkers for urological and other diseases. Moreover, the urine collection is easy and noninvasive and urinary exosomes give information about renal and systemic organ systems. Accordingly, multiple studies on proteomic characterization of urinary exosomes in health and disease have been published. However, no systematic analysis of their glycoproteomic profile has been carried out to date, whereas a conserved glycan signature has been found for exosomes from urine and other sources including T cell lines and human milk. Here, we have enriched and identified the N-glycopeptides from these vesicles. These enriched N-glycopeptides were solved for their peptide sequence, glycan composition, structure, and glycosylation site using collision-induced dissociation MS/MS (CID-tandem MS) data interpreted by a publicly available software GlycopeptideId. Released glycans from the same sample was also analyzed with MALDI-MS. We have identified the N-glycoproteome of urinary exosomes. In total 126 N-glycopeptides from 51 N-glycosylation sites belonging to 37 glycoproteins were found in our results. The peptide sequences of these N-glycopeptides were identified unambiguously and their glycan composition (for 125 N-glycopeptides) and structures (for 87 N-glycopeptides) were proposed. A corresponding glycomic analysis with released N-glycans was also performed. We identified 66 unique nonmodified N-glycan compositions and in addition 13 sulfated/phosphorylated glycans were also found. This is the first systematic analysis of N-glycoproteome of urinary exosomes.

Glycosylation patterns of kidney proteins differ in rat diabetic nephropathy.

Diabetic nephropathy often progresses to end-stage kidney disease and, ultimately, to renal replacement therapy. Hyperglycemia per se is expected to have a direct impact on the biosynthesis of N- and O-linked glycoproteins. This study aims to establish the link between protein glycosylation and progression of experimental diabetic kidney disease using orthogonal methods. Kidneys of streptozotocin-diabetic and control rats were harvested at three different time points post streptozotocin injection. A panel of 12 plant lectins was used in the screening of lectin blots. The lectins UEAI, PHA-E, GSI, PNA, and RCA identified remarkable disease-associated differences in glycoprotein expression. Lectin affinity chromatography followed by mass spectrometric analyses led to the identification of several glycoproteins involved in salt-handling, angiogenesis, and extracellular matrix degradation. Our data confirm a substantial link between glycosylation signature and diabetes progression. Furthermore, as suggested by our findings on dipeptidyl peptidase-IV, altered protein glycosylation may reflect changes in biochemical properties such as enzymatic activity. Thus, our study demonstrates the unexplored potential of protein glycosylation analysis in the discovery of molecules linked to diabetic kidney disease.

Ezrin is down-regulated in diabetic kidney glomeruli and regulates actin reorganization and glucose uptake via GLUT1 in cultured podocytes.

Diabetic nephropathy is a complication of diabetes and a major cause of end-stage renal disease. To characterize the early pathophysiological mechanisms leading to glomerular podocyte injury in diabetic nephropathy, we performed quantitative proteomic profiling of glomeruli isolated from rats with streptozotocin-induced diabetes and controls. Fluorescence-based two-dimensional difference gel electrophoresis, coupled with mass spectrometry, identified 29 differentially expressed spots, including actin-binding protein ezrin and its interaction partner, NHERF2, which were down-regulated in the streptozotocin group. Knockdown of ezrin by siRNA in cultured podocytes increased glucose uptake compared with control siRNA-transfected cells, apparently by increasing translocation of glucose transporter GLUT1 to the plasma membrane. Knockdown of ezrin also induced actin remodeling under basal conditions, but reduced insulin-stimulated actin reorganization. Ezrin-dependent actin remodeling involved cofilin-1 that is essential for the turnover and reorganization of actin filaments. Phosphorylated, inactive cofilin-1 was up-regulated in diabetic glomeruli, suggesting altered actin dynamics. Furthermore, IHC analysis revealed reduced expression of ezrin in the podocytes of patients with diabetes. Our findings suggest that ezrin may play a role in the development of the renal complication in diabetes by regulating transport of glucose and organization of the actin cytoskeleton in podocytes.

Equine mesenchymal stem cell-derived extracellular vesicle productivity but not overall yield is improved via 3-D culture with chemically defined media.

OBJECTIVE: Mesenchymal stem cell (MSC) extracellular vesicles (EVs) have emerged as a biotherapeutic for osteoarthritis; however, manufacturing large quantities is not practical using traditional monolayer (2-D) culture. We aimed to examine the effects of 3-D and 2-D culture 2 types of media: Dulbecco modified Eagle medium and a commercially available medium (CM) on EV yield. ANIMALS: Banked bone marrow-derived MSCs (BM-MSCs) from 6 healthy, young horses were used. METHODS: 4 microcarriers (collagen-coated polystyrene, uncoated polystyrene, collagen-coated dextran, and uncoated dextran) were tested in static and bioreactor cultures, and the optimal microcarrier was chosen. The BM-MSCs were inoculated into a bioreactor with collagen-coated dextran microcarriers at 5,000 cells/cm2 or onto culture dishes at 4,000 cells/cm2 in either Dulbecco modified Eagle medium or CM media. Supernatants were obtained for metabolite and pH analysis. The BM-MSCs were expanded until confluent (2-D) or for 7 days (3-D) when the 48-hour EV collection period commenced using EV-depleted media. Extracellular vesicles were isolated and characterized via nanoparticle tracking analysis, Western blot, transmission electron microscopy, and protein quantification. The BM-MSCs were harvested, quantified, and immunophenotyped. RESULTS: The number of EVs isolated was not improved by 3-D culture or CM media, however, the CM 3-D condition improved the number of EVs produced per BM-MSC over the CM 2-D condition (mean ± SD: 306 ± 99 vs 37 ± 22, respectively). Glucose decreased and lactate and ammonium accumulated in 3-D culture. Surface markers of stemness exhibited reduced expression in 3-D culture. CLINICAL RELEVANCE: Optimization of our 3-D culture methods could improve BM-MSC expansion and thus EV yield.

Expression of the αVß3 integrin affects prostate cancer sEV cargo and density and promotes sEV pro-tumorigenic activity in vivo through a GPI-anchored receptor, NgR2.

It is known that small extracellular vesicles (sEVs) are released from cancer cells and contribute to cancer progression via crosstalk with recipient cells. We have previously reported that sEVs expressing the αVß3 integrin, a protein upregulated in aggressive neuroendocrine prostate cancer (NEPrCa), contribute to neuroendocrine differentiation (NED) in recipient cells. Here, we examine the impact of αVß3 expression on sEV protein content, density and function. sEVs used in this study were isolated by iodixanol density gradients and characterized by nanoparticle tracking analysis, immunoblotting and single vesicle analysis. Our proteomic profile of sEVs containing αVß3 shows downregulation of typical effectors involved in apoptosis and necrosis and an upregulation of tumour cell survival factors compared to control sEVs. We also show that the expression of αVß3 in sEVs causes a distinct reposition of EV markers (Alix, CD81, CD9) to a low-density sEV subpopulation. This low-density reposition is independent of extracellular matrix (ECM) protein interactions with sEVs. This sEV subset contains αVß3 and an αVß3 downstream effector, NgR2, a novel marker for NEPrCa. We show that sEVs containing αVß3 are loaded with higher amounts of NgR2 as compared to sEVs that do not express αVß3. Mechanistically, we demonstrate that sEVs containing NgR2 do not affect the sEV marker profile, but when injected in vivo intratumorally, they promote tumour growth and induce NED. We show that sEVs expressing NgR2 increase the activation of focal adhesion kinase (FAK), a known promoter of cancer cell proliferation, in recipient cells. We also show that NgR2 mimics the effect of sEVs containing αVß3 since it displays increased growth of NgR2 transfectants in vivo, as compared to control cells. Overall, our results describe the changes that occur in cargo, density and functions of cancer cell-derived sEVs containing the αVß3 integrin and its effector, NgR2, without affecting the sEV tetraspanin profiles.

Recovery of urinary nanovesicles from ultracentrifugation supernatants.

BACKGROUND: Urinary vesicles represent a newly established source of biological material, widely considered to faithfully represent pathological events in the kidneys and the urogenital epithelium. The majority of currently applied isolation protocols involve cumbersome centrifugation steps to enrich vesicles from urine. To date, the efficiency of these approaches has not been investigated with respect to performing quantitative and qualitative analyses of vesicle populations in the pellet and supernatant (SN) fractions. METHODS: After the series of differential centrifugations, the final SN was reduced to one-twentieth of the original volume by ammonium sulphate precipitation, with the precipitate pellet subjected to another round of differential centrifugations. Electron microscopy, dynamic light scattering and western blot analysis were used to characterize the vesicles present in individual fractions of interest. RESULTS: Pellets obtained after the second set of centrifugations at 200 000 g revealed the presence of vesicles which share a common marker profile, but with distinct differences from those seen in the initial 200 000 g pellet used as the reference. This suggests an enrichment of previously uncharacterized urinary vesicles still in solution after the initial centrifugation steps. Analysis of protein yields recovered post-ultracentrifugation revealed an additional 40% of vesicles retained from the SN. Moreover, these structures showed a formidable resistance to harsh treatments (e.g. 95% ammonium sulphate saturation, hypotonic dialysis, 0.3 M sodium hydroxide). CONCLUSIONS: Methods which employ differential centrifugations of native urine are remarkably ineffective and may lose a substantial population of biologically important vesicle species.

Diabetes with kidney injury may change the abundance and cargo of urinary extracellular vesicles.

Background: Urinary extracellular vesicles (uEVs) are derived from epithelia facing the renal tubule lumen in the kidney and urogenital tract; they may carry protein biomarkers of renal dysfunction and structural injury. However, there are scarce studies focusing on uEVs in diabetes with kidney injury. Materials and methods: A community-based epidemiological survey was performed, and the participants were randomly selected for our study. uEVs were enriched by dehydrated dialysis method, quantified by Coomassie Bradford protein assay, and adjusted by urinary creatinine (UCr). Then, they identified by transmission electron microscopy (TEM), nanoparticle track analysis (NTA), and western blot of tumor susceptibility gene 101. Results: Decent uEVs with a homogeneous distribution were finally obtained, presenting a membrane-encapsulated structure like cup-shaped or roundish under TEM, having active Brownian motion, and presenting the main peak between 55 and 110 nm under NTA. The Bradford protein assay showed that the protein concentrations of uEVs were 0.02 ± 0.02, 0.04 ± 0.05, 0.05 ± 0.04, 0.07 ± 0.08, and 0.11 ± 0.15 µg/mg UCr, respectively, in normal controls and in prediabetes, diabetes with normal proteinuria, diabetes with microalbuminuria, and diabetes with macroproteinuria groups after adjusting the protein concentration with UCr by calculating the vesicles-to-creatinine ratio. Conclusion: The protein concentration of uEVs in diabetes with kidney injury increased significantly than the normal controls before and after adjusting the UCr. Therefore, diabetes with kidney injury may change the abundance and cargo of uEVs, which may be involved in the physiological and pathological changes of diabetes.

Extracellular vesicles and insulin-mediated vascular function in metabolic syndrome.

Metabolic Syndrome (MetS) raises cardiovascular disease risk. Extracellular vesicles (EVs) have emerged as important mediators of insulin sensitivity, although few studies on vascular function exist in humans. We determined the effect of insulin on EVs in relation to vascular function. Adults with MetS (n = 51, n = 9 M, 54.8 ± 1.0 years, 36.4 ± 0.7 kg/m2 , ATPIII: 3.5 ± 0.1 a.u., VO2 max: 22.1 ± 0.6 ml/kg/min) were enrolled in this cross-sectional study. Peripheral insulin sensitivity (M-value) was determined during a euglycemic clamp (40 mU/m2 /min, 90 mg/dl), and blood was collected for EVs (CD105+, CD45+, CD41+, TX+, and CD31+; spectral flow cytometry), inflammation, insulin, and substrates. Central hemodynamics (applanation tonometry) was determined at 0 and 120 min via aortic waveforms. Pressure myography was used to assess insulin-induced arterial vasodilation from mouse 3rd order mesenteric arteries (100-200 µm in diameter) at 0.2, 2 and 20 nM of insulin with EVs from healthy and MetS adults. Adults with MetS had low peripheral insulin sensitivity (2.6 ± 0.2 mg/kg/min) and high HOMA-IR (4.7 ± 0.4 a.u.) plus Adipose-IR (13.0 ± 1.3 a.u.). Insulin decreased total/particle counts (p < 0.001), CD45+ EVs (p = 0.002), AIx75 (p = 0.005) and Pb (p = 0.04), FFA (p < 0.001), total adiponectin (p = 0.006), ICAM (p = 0.002), and VCAM (p = 0.03). Higher M-value related to lower fasted total EVs (r = -0.40, p = 0.004) while higher Adipose-IR associated with higher fasted EVs (r = 0.42, p = 0.004) independent of VAT. Fasting CD105+ and CD45+ derived total EVs correlated with fasting AIx75 (r = 0.29, p < 0.05) and Pb (r = 0.30, p < 0.05). EVs from MetS participants blunted insulin-induced vasodilation in mesenteric arteries compared with increases from healthy controls across insulin doses (all p < 0.005). These data highlight EVs as potentially novel mediators of vascular insulin sensitivity and disease risk.

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles.

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field.

Rigorous characterization of urinary extracellular vesicles (uEVs) in the low centrifugation pellet - a neglected source for uEVs.

Urinary extracellular vesicles (uEVs) provide bio-markers for kidney and urogenital diseases. Centrifugation is the most common method used to enrich uEVs. However, a majority of studies to date have focused on the ultracentrifugation pellet, potentially losing a novel source of important biomarkers that could be obtained at lower centrifugation. Thus, the aim of this study is to rigorously characterize for the first time uEVs in the low speed pellet and determine the minimal volume of urine required for proteomic analysis (≥9.0 mL urine) and gene ontology classification identified 75% of the protein as extracellular exosomes. Cryo-Transmission Electron Microscopy (≥3.0 mL urine) provided evidence of a heterogeneous population of EVs for size and morphology independent of uromodulin filaments. Western blot detected several specific uEV kidney and EV markers (≥4.5 mL urine per lane). microRNAs quantification by qPCR was possible with urine volume as low as 0.5 mL. Particle enumeration with tunable resistive pulse sensing, nano particles tracking analysis and single EV high throughput imaging flow cytometry are possible starting from 0.5 and 3.0 mL of urine respectively. This work characterizes a neglected source of uEVs and provides guidance with regard to volume of urine necessary to carry out multi-omic studies and reveals novel aspects of uEV analysis such as autofluorescence of podocyte origin.

Circulating Extracellular Vesicles in Normotension Restrain Vasodilation in Resistance Arteries.

Extracellular vesicles (EVs) have been described as novel biomarkers and bioactivators in vascular dysfunction in hypertension. However, the mechanism(s) by which EVs affect vascular function is unknown. To examine the effects of EVs on endothelial-dependent vasodilation (acetylcholine), we isolated circulating EVs from platelet-poor plasma using a low centrifugation speed (17 000g) and mesenteric resistance arteries from 12-week-old normotensive WKYs (Wistar-Kyoto rats) and SHRs (spontaneously hypertensive rats). Arteries were cannulated on a pressure myograph, and EVs were added to the vessel lumen and circulating bath. We found that circulating EVs from normotensive WKY reduced vasodilation of normotensive WKY arteries but had no effect on hypertensive SHR arteries. In contrast, EVs from hypertensive SHR failed to reduce vasodilation of arteries from both WKY and SHR. The restraining effect on vasodilation by EVs from normotensive WKY may be mediated by inhibition of eNOS (endothelial NO synthase), as addition of L-nitro-arginine methyl ester did not provide any additive effect. Moreover, circulating EVs from normotensive 6-week-old SHR-an age where SHRs have not yet developed hypertension-had similar restraining effect on vasodilation. In addition, delipidation of EVs did not alter the restraining effect of EVs from WKY but did restore the restraining effect of EVs from SHR. Finally, EVs from normotensive humans also restrained vasodilation of normotensive mouse arteries-an effect not observed in EVs from hypertensive humans. Taken together, our data support a vasoactive role of EVs that is altered in hypertension.

Interval Exercise Lowers Circulating CD105 Extracellular Vesicles in Prediabetes.

BACKGROUND: Extracellular vesicles (EV) are purported to mediate type 2 diabetes and CVD risk and development. Physical activity and a balanced diet reduce disease risk, but no study has tested the hypothesis that short-term interval (INT) training would reduce EV compared with continuous (CONT) exercise in adults with prediabetes. METHODS: Eighteen obese adults (age, 63.8 ± 1.5 yr; body mass index, 31.0 ± 1.3 kg·m) were screened for prediabetes using American Diabetes Association criteria (75 g oral glucose tolerance test). Subjects were randomized to INT (n = 10, alternating 3-min intervals at 90% and 50% HRpeak, respectively) or CONT (n = 8, 70% HRpeak) training for 12 supervised sessions over 13 d for 60 min·d. Cardiorespiratory fitness (V˙ O2peak), weight (kg), as well as ad libitum dietary intake were assessed and arterial stiffness (augmentation index via applanation tonometry) was calculated using total AUC during a 75-g oral glucose tolerance test performed 24 h after the last exercise bout. Total EV, platelet EV (CD31/CD41), endothelial EV (CD105; CD31/ CD41), platelet endothelial cell adhesion molecule (PECAM) (CD31), and leukocyte EV (CD45; CD45/CD41) were analyzed via imaging flow cytometry preintervention/postintervention. RESULTS: The INT exercise increased V˙O2peak (P = 0.04) compared with CONT training. Although training had no effect on platelet or leukocyte EV, INT decreased Annexin V- endothelial EV CD105 compared with CONT (P = 0.04). However, after accounting for dietary sugar intake, the intensity effect was lost (P = 0.18). Increased ad libitum dietary sugar intake after training was linked to elevated AV+ CD105 (r = 0.49, P = 0.06) and AV- CD45 (r = 0.59, P = 0.01). Nonetheless, increased V˙O2peak correlated with decreased AV+ CD105 (r = -0.60, P = 0.01). CONCLUSIONS: Interval exercise training decreases endothelial-derived EV in adults with prediabetes. Although increased sugar consumption may alter EV after a short-term exercise intervention, fitness modifies EV count.

A miRNA signature in endothelial cell-derived extracellular vesicles in tumor-bearing mice.

Extracellular vesicles (EVs) play important roles in tumor progression by altering immune surveillance, promoting vascular dysfunction, and priming distant sites for organotropic metastases. The miRNA expression patterns in circulating EVs are important diagnostic tools in cancer. However, multiple cell types within the tumor microenvironment (TME) including cancer cells and stromal cells (e.g. immune cells, fibroblasts, and endothelial cells, ECs) contribute to the pool of circulating EVs. Because EVs of different cellular origins have different functional properties, auditing the cargo derived from cell type-specific EVs in the TME is essential. Here, we demonstrate that a murine EC lineage-tracing model (Cdh5-CreERT2:ZSGreenl/s/l mice) can be used to isolate EC-derived extracellular vesicles (EC-EVs). We further show that purified ZSGreen+ EVs express expected EV markers, they are transferable to multiple recipient cells, and circulating EC-EVs from tumor-bearing mice harbor elevated levels of specific miRNAs (e.g. miR-30c, miR-126, miR-146a, and miR-125b) compared to non tumor-bearing counterparts. These results suggest that, in the tumor setting, ECs may systemically direct the function of heterotypic cell types either in the circulation or in different organ micro-environments via the cargo contained within their EVs.

An Oral Glucose Load Decreases Postprandial Extracellular Vesicles in Obese Adults with and without Prediabetes.

Although extracellular vesicles (EVs) are a novel biomediator of type 2 diabetes (T2D) and cardiovascular disease (CVD), the effects of hyperglycemia on EVs in humans is unknown. We tested the hypothesis that a 75-g oral glucose tolerance test (OGTT) would promote changes in EVs in relation to CVD risk. Twenty-five obese adults (Age: 52.4 ± 3.2 year, BMI: 32.5 ± 1.2 kg/m²) were screened for normal glucose tolerance (NGT, n = 8) and prediabetes (PD, n = 17) using American Diabetes Association criteria (75 g OGTT and/or HbA1c). Body composition (bioelectrical impedance) and fitness (VO2peak) were measured. Arterial stiffness (augmentation index; AIx) was measured at 0, 60- and 120-min while insulin, glucose, and free fatty acids were evaluated every 30 min during the OGTT to assess CVD risk. Annexin V positive (AV+) and Annexin V negative (AV-) total EVs, platelet EVs (CD31⁺/CD41⁺; CD41⁺), leukocyte EVs (CD45⁺; CD45⁺/CD41-), platelet endothelial cell adhesion molecule (PECAM) (CD31⁺) and endothelial EVs (CD 31⁺/CD41-; CD105⁺) were collected at 0 and 120 min. There were no differences in age, BMI, or body fat between NGT and PD (all P > 0.63). Total EVs, AV+ CD31+ (PECAM), and AV+ CD31⁺/CD41- (endothelial) EVs decreased after the OGTT (P ≤ 0.04). Circulating insulin at 2-h correlated with elevated post-prandial AV- CD45⁺ (r = 0.48, P = 0.04) while arterial stiffness related to reduced total EVs (r = -0.49, P = 0.03) and AV+CD41⁺ (platelet) (r = -0.52, P = 0.02). An oral glucose load lowers post-prandial total, platelet, and endothelial EVs in obese adults with NGT and prediabetes in relation to CVD risk.