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

Abstract

Medium/large extracellular vesicles (m/lEVs) are released by most cell types and are involved in multiple basic biological processes. Analysis of m/lEV levels in blood or urine may help unravel pathophysiological findings in many diseases. However, it remains unclear how many naturally-occurring m/lEV subtypes exist as well as how their characteristics and functions differ from one another. Here, we identified m/lEVs pelleted from plasma and urine samples by differential centrifugation and showed by flow cytometry that they typically possessed diameters between 200 nm and 800 nm. Using proteomic profiling, we identified several proteins involved in m/lEV biogenesis including adhesion molecules, peptidases and exocytosis regulatory proteins. In healthy human plasma, we could distinguish m/lEVs derived from platelets, erythrocytes, monocytes/macrophages, T and B cells, and vascular endothelial cells using various surface antigens. m/lEVs derived from erythrocytes and monocytes were Annexin V positive. In urine, 50% of m/lEVs were Annexin V negative but contained various membrane peptidases derived from renal tubular villi. Urinary m/lEVs, but not plasma m/lEVs, showed peptidase activity. The method we have developed to characterize cellderived m/lEVs suggests the possibility of clinical applications.

Where Metabolism Meets Senescence: Focus on Endothelial Cells

“…To address this issue, the field is moving toward more specific approaches allowing the deconvolution of complex circulating signatures, including the application of machine learning tools and the study of EVs (Heitzer et al, 2019). A growing body of evidence is focusing on the characterization of endothelial EVs isolated from plasma (Igami et al, 2019;Mork et al, 2019;Oliveira et al, 2019;Utermohlen et al, 2019). Even if a consensus on the methods for isolation and characterization of these EVs is far from being reached, it is important to remark that the profiling of the cargo of circulating EVs sorted according to the parent cells allows enhanced specificity compared to single molecular markers in the blood.…”

Hijacking Endocytosis and Autophagy in Extracellular Vesicle Communication: Where the Inside Meets the Outside

“…For instance, PS on the EV surface may interact with the PS-ligand annexin-V enriched in membranes of early and late endocytic organelles of macrophages ( Diakonova et al, 1997 ). In an opposite manner, PS on the luminal side of endocytic membranes ( Matsudaira et al, 2017 ) binds to annexin-V present on the surface of apoptotic bodies ( Igami et al, 2020 ). Cholesterol is a key component of membrane organization and fusion events ( Yang et al, 2016 ).…”

Extracellular vesicle-based therapy for amyotrophic lateral sclerosis

“…[ 16 51 ] Finally, annexin V, C2b, and thrombospondin have been delineated in apoptotic bodies. [ 52 53 54 ] To more effectively distinguish EV subtypes, a ratio comparing the number of a particular biomarker with the others in the vesicles should be determined. [ 17 ] Additionally, proteomic studies and analysis of RNA in EVs are key factors to delineate the molecular cargo of EVs and may aid in elucidating the EV mechanisms in ALS.…”