Abstract
Exosome secretion by cells is a complex, poorly understood process. Studies of exosomes would be facilitated by a method for increasing their production and release. Here, we present a method for stimulating the secretion of exosomes. Cultured cells were treated or not with sodium iodoacetate (IAA; glycolysis inhibitor) plus 2,4-dinitrophenol (DNP; oxidative phosphorylation inhibitor). Exosomes were isolated by size-exclusion chromatography and their morphology, size, concentration, cargo components and functional activity were compared. IAA/DNP treatment (up to 10 µM each) was non-toxic and resulted in a 3 to 16-fold increase in exosome secretion. Exosomes from IAA/DNP-treated or untreated cells had similar biological properties and functional effects on endothelial cells (SVEC4-10). IAA/DNP increased exosome secretion from mouse organ cultures, and in vivo injections enhanced the levels of circulating exosomes. IAA/DNP decreased ATP levels (p < 0.05) in cells. A cell membrane-permeable form of 2′,3′-cAMP and 3′-AMP mimicked the potentiating effects of IAA/DNP on exosome secretion. In cells lacking 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase; an enzyme that metabolizes 2′,3′-cAMP into 2′-AMP), effects of IAA/DNP on exosome secretion were enhanced. The IAA/DNP combination is a powerful stimulator of exosome secretion, and these stimulatory effects are, in part, mediated by intracellular 2′,3′-cAMP.
Highlights
Carry addressed instructions to the potential recipient cells[4]
There is a need for standardizing and optimizing exosome production by cultured cell lines, and while we have previously described methods for achieving such optimization[11,12], here we report a newly developed procedure for increasing exosome production in vitro and in vivo using a combination of two biochemical agents, sodium iodoacetate (IAA; glycolysis inhibitor) and 2,4-dinitrophenol (DNP; oxidative phosphorylation inhibitor)
Because cyclic nucleotide 3′-phosphodiesterase (CNPase) metabolizes intracellular 2′,3′-cAMP, we examined the www.nature.com/scientificreports release of exosomes induced by either 8-Br-2′,3′-cAMP or IAA/DNP in Preglomerular vascular smooth muscle cells (PGVSMCs) obtained from CNPase+/+ versus CNPase −/− rats
Summary
Carry addressed instructions to the potential recipient cells[4]. secreted and circulating exosomes are looked upon as a liquid biopsy, and their characteristics, cargos of proteins and nucleic acids and their identity with the parent cells have been of great interest. Exosomes are in demand as potential non-invasive biomarkers and as a delivery system of messages that can be transferred to or incorporated into the exosome cargo[5]. Exosome cargos avoid degradation or loss during delivery to distant sites[10]. Due to these characteristics, exosomes are considered to be favourable component of vaccines. There is a need for standardizing and optimizing exosome production by cultured cell lines, and while we have previously described methods for achieving such optimization[11,12], here we report a newly developed procedure for increasing exosome production in vitro and in vivo using a combination of two biochemical agents, sodium iodoacetate (IAA; glycolysis inhibitor) and 2,4-dinitrophenol (DNP; oxidative phosphorylation inhibitor)
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