Optimal Isolation Method of Small Extracellular Vesicles from Rat Plasma

Abstract

Small extracellular vesicles (sEVs) mediate cell–to–cell communication. We recently reported that circulating sEVs regulate systolic blood pressure in an animal model of human systemic hypertension. However, the underlying mechanisms still remain to be elucidated. As the first step for detailed analyses, we sought to increase the yield and purity of sEVs isolated from rat plasma. We compared the concentration and size distribution of sEVs as well as protein expression of the sEV marker and contaminants among plasma sEVs isolated by the ultracentrifugation (UC) method, the precipitation with polyethylene-glycol and ultracentrifugation (PEG-UC) method, or the precipitation with polyethylene-glycol (PEG) method. Effects of anticoagulants were also examined. The total concentration of plasma sEVs isolated by the PEG or PEG-UC method was much higher than that of the UC method. In the plasma sEVs isolated by the PEG-UC method, contaminating proteins were lower, while the protein expression of certain sEV markers was higher than that of the PEG method. There was no significant difference in total concentration or protein expression of sEV markers in sEVs isolated from rat plasma treated with three different anticoagulants (heparin, ethylenediaminetetraacetic acid, or acid citrate dextrose buffer) by the PEG-UC method. We, for the first time, determined that the PEG-UC method was optimal for sEV isolation from rat plasma.

Small extracellular vesicles from rat plasma promote migration and proliferation of vascular smooth muscle cells

“…sEV isolation from plasma sEV were isolated from plasma of WKY (WsEV) and SHR (SsEV) by the precipitation with polyethylene-glycol and ultracentrifugation [27]. WKY and SHR (6-week-old) were deeply anesthetized with urethane (Sigma-Aldrich, St. Louis, MO, USA) (1.5 g/kg, i.p.…”

“…The concentration and size distribution of the isolated vesicles were measured by a tunable resistive pulse sensing (TRPS) method using a qNANO instruments with an NP150 nanopore at 46.0-47.0 mm stretch (IZON Science, Christchurch, New Zealand), which was coated with a TRPS Reagent Kit (IZON Science) for the prevention of sEV adhesion according to a manufacturer's instruction [27]. Raw data were standardized by carboxylated-polystyrene particles with a known diameter of 110 nm.…”

“…The protein expression in plasma sEV was determined by Western blotting as previously described [27]. Protein lysates of sEV were extracted using a radio-immunoprecipitation assay buffer containing 20 mM Tris-HCl (pH7.5), 150 mM NaCl, 10 mM MgCl 2 , 1% Triton-X, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% protease inhibitor mixture, and 1% phosphatase inhibitor mixture (Nacalai Tesque) (4°C, 10 min).…”

Plasma small extracellular vesicles in hypertensive rats impair reactivity of isolated blood vessels

“…Particle distribution and concentration of the isolated sEV were measured by a tunable resistive pulse sensing (TRPS) method using a qNANO instrument with an NP150 nanopore at 46.5-47.5 mm stretch (IZON Science, Christchurch, New Zealand) according to the manufacturer's instruction as previously reported [15,16].…”

Gonadotropin-releasing hormone increases the release of extracellular vesicles from gonadotropes

“…Ultrathin sections were cut using an Ultracut N (Reichert-Nissei, Wein, Austria), stained using uranyl acetate followed by lead citrate and examined using a Hitachi H-7650 transmission electron microscope (Hitachi Ltd, Tokyo, Japan). The particulate fractions were fixed and stained with phosphotungstic acid on a carbon-coated copper grid and observed by using TEM (80 MeV, × 15000) as reported previously (Otani et al, 2019).…”