The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution and functions. By employing asymmetric flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90–120 nm; small exosome vesicles, Exo-S, 60–80 nm) and discovered an abundant population of non-membranous nanoparticles termed ‘exomeres’ (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins as well as specific pathways, such as glycolysis and mTOR signalling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signalling pathways, respectively. Exo-S, Exo-L and exomeres each had unique N-glycosylation, protein, lipid, DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating extracellular vesicles and addressing the complexities of heterogeneous nanoparticle subpopulations.

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high‐throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture “what the community needs in a tool”. Here, we describe a novel community‐driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.

Background: Galectin‐3 (Gal‐3) participates in different mechanisms involved in atherothrombosis, such as inflammation, proliferation, or macrophage chemotaxis. Thus, there have been committed intensive efforts to elucidate the function of Gal‐3 in cardiovascular ( CV ) diseases. The role of Gal‐3 as a circulating biomarker has been demonstrated in patients with heart failure, but its importance as a biomarker in atherothrombosis is still unknown. Methods and Results: Because Gal‐3 is involved in monocyte‐to‐macrophage transition, we used fresh isolated monocytes and the in vitro model of macrophage differentiation of THP ‐1 cells stimulated with phorbol myristate acetate ( PMA) . Gal‐3 release is increased by PMA in human monocytes and macrophages, a process involving exosomes and regulated by reactive oxygen species/ NADPH oxidase activity. In asymptomatic subjects (n=199), Gal‐3 plasma levels are correlated with NADPH oxidase activity in peripheral blood mononuclear cells ( r =0.476; P <0.001) and carotid intima‐media thickness ( r =0.438; P <0.001), a surrogate marker of atherosclerosis. Accordingly, Gal‐3 plasma concentrations are increased in patients with carotid atherosclerosis (n=158), compared to control subjects (n=115; 14.3 [10.7 to 16.9] vs. 10.4 [8.6 to 12.5] ng/ mL ; P <0.001). Finally, on a 5‐year follow‐up study in patients with peripheral artery disease, Gal‐3 concentrations are significantly and independently associated with an increased risk for CV mortality (hazard ratio=2.24, 95% confidence interval: 1.06 to 4.73, P <0.05). Conclusions: Gal‐3 extracellular levels could reflect key underlying mechanisms involved in atherosclerosis etiology, development, and plaque rupture, such as inflammation, infiltration of circulating cells and oxidative stress. Moreover, circulating Gal‐3 concentrations are associated with clinical outcomes in patients with atherothrombosis.

Urinary extracellular vesicles provide a novel source for valuable biomarkers for kidney and urogenital diseases: Current isolation protocols include laborious, sequential centrifugation steps which hampers their widespread research and clinical use. Furthermore, large individual urine sample volumes or sizable target cohorts are to be processed (e.g. for biobanking), the storage capacity is an additional problem. Thus, alternative methods are necessary to overcome such limitations. We have developed a practical vesicle isolation technique to yield easily manageable sample volumes in an exceptionally cost efficient way to facilitate their full utilization in less privileged environments and maximize the benefit of biobanking. Urinary vesicles were isolated by hydrostatic dialysis with minimal interference of soluble proteins or vesicle loss. Large volumes of urine were concentrated up to 1/100 of original volume and the dialysis step allowed equalization of urine physico-chemical characteristics. Vesicle fractions were found suitable to any applications, including RNA analysis. In the yield, our hydrostatic filtration dialysis system outperforms the conventional ultracentrifugation-based methods and the labour intensive and potentially hazardous step of ultracentrifugations are eliminated. Likewise, the need for trained laboratory personnel and heavy initial investment is avoided. Thus, our method qualifies as a method for laboratories working with urinary vesicles and biobanking.