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.
Highlights
Cells release lipid-bilayer-capsuled particles containing various molecules, such as proteins, DNA, mRNA, small RNA, and others, into extracellular fluid [1,2]
We reported that plasma sEVs in a spontaneously hypertensive rat (SHR), an animal model of human systemic hypertension, regulate systolic blood pressure [8]
The contaminations of large extracellular vesicles (EVs) marker protein (α-actinin-4) and plasma albumin in sEVs isolated by the polyethylene-glycol and ultracentrifugation (PEG-UC) method were much lower than that by the PEG method (Figure 3f,g)
Summary
Cells release lipid-bilayer-capsuled particles containing various molecules, such as proteins, DNA, mRNA, small RNA, and others, into extracellular fluid [1,2]. The contaminations of large EV marker protein (α-actinin-4) and plasma albumin in sEVs isolated by the PEG-UC method were much lower than that by the PEG method (Figure 3f,g) These results indicate that a washing procedure by ultracentrifugation is effective to eliminate contaminants and to enhance the purity of isolated sEVs as previously reported [15,23]. Large EVs in the plasma were directly detected by a flow cytometry, while sEVs were not detected This is one possible explanation for the discrepancy, and we believe that our results represent a more accurate population of plasma sEVs. In sEVs from the ACD-treated plasma, the total protein concentration was significantly higher than that from the EDTA-treated plasma, while plasma albumin expression was significantly lower compared with other plasma preparations (Figure 5a,g). Our findings could contribute to better understanding mechanisms underlying various diseases, including hypertension, diabetes, obesity, and others, mediated by circulating sEVs, especially in rat models
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