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Characterization of exosomes isolated from cultured vascular endothelial and smooth muscle cells
Author(s) -
Boyer Michael,
Baggett Ariele,
Scalia Rosario,
Eguchi Satoru,
Rizzo Victor
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.837.7
Subject(s) - microvesicles , exosome , vascular smooth muscle , microbiology and biotechnology , vesicle , chemistry , endosome , cell , nanoparticle tracking analysis , centrifugation , adventitia , biophysics , biology , anatomy , biochemistry , membrane , smooth muscle , endocrinology , microrna , gene
Exosomes are small nanovesicles derived from endosomal compartments termed multivesicular bodies. Upon biogenesis, the multivesicular bodies fuse with the plasma membrane, releasing the exosomes, which contain functional nucleic acids and proteins. Despite their well‐documented existence, not much is known about vascular exosomes. The basic architecture of a blood vessel consists of the intima, media, and adventitia. The luminal surface of the intima consists of a confluent monolayer of endothelial cells (ECs), while the medial layer is characterized by presence of vascular smooth muscle cells (VSMCs). We characterized the exosomes released by these cell types in culture in order to better understand the packaging of these vesicles and their capacity to participate in potential cell‐to‐cell communication. Rat aortic ECs exosomes were generated in serum‐free media for 48 hours while rat aortic VSMC exosomes were generated in serum‐free media for 72 hours. Media harvested from both was ultracentrifuged at 10,000×g to remove microparticles followed by centrifugation of the supernatant at 100,000×g to pellet exosomes. Exosome pellet was washed once using PBS and spun at 100,000×g. Transmission electron microscopic analysis of VSMC exosomes confirmed particle morphology with diameters ranging from 60–150 nm. Quantitative evaluations of particle size and concentration were further performed using NTA NanoSight. Average sizes of EC exosomes and VSMC exosomes were 161.9±49.9nm and 176.7±66.4nm, respectively. The concentrations of EC and VSMC exosomes were 2.24×10 10 particles/mL/day and 7.63×10 10 particles/mL/day, respectively. Western blotting analysis was performed to characterize common and specific markers for vascular exosomes. We found common exosome makers in both ECs and VSMCs, such as flotillin‐1 and CD81. ECs specifically expressed VE‐cadherin and TSG‐101 whereas VSMC expressed syntenin‐1. From our data, we conclude that exosomes are constitutively released from these cell types and play a potential role in vascular cell‐to‐cell communication. The methods herein provide useful information regarding the characterization of vascular exosomes in cardiovascular physiology.