Autocrine role of senescent cardiac fibroblasts-derived extracellular vesicles.

Cellular senescence is a highly stable state associated with cell cycle arrest, that is elicited in response to various stresses. The accumulation of senescent cells in tissues drives age-related diseases. Recent studies have shown that the cellular senescence enhances an extracellular vesicles (EV) secretion. EV are lipid-bilayer-capsuled particles released by various cells mediating cell-to-cell communication. It was recently reported that EV secreted by the senescent cells had several functions such as cancer cell proliferation and immune cell activation. In the present study, we investigated whether senescent cardiac fibroblasts-derived EV play an autocrine/paracrine role in the heart cells. Neonatal rat cardiac fibroblasts (NRCFs) were treated with doxorubicin (DOX) to induce cellular senescence. EV were isolated from NRCFs culture media. The vehicle-treated NRCFs-derived EV (D0-EV, 72 hr) increased a living cell number in NRCFs, which was attenuated by DOX (1,000 nM)-treated NRCFs-derived EV (D103-EV, 72 hr). While D0-EV did not affect protein concentration in NRCFs, D103-EV decreased it. Furthermore, D103-EV significantly increased a ratio of microtubule-associated protein 1 light chain 3 (LC3)-II to LC3-I in NRCFs, indicating an induction of autophagy. In addition, D103-EV increased phosphorylation of adenosine monophosphate-activated kinase (AMPK) α in NRCFs. In neonatal rat cardiomyocytes, however, NRCFs-derived EV (72 hr) had no effect on the living cell number, protein concentration, and ratio of LC3-II to LC3-I. In conclusion, we for the first time revealed that DOX-induced senescent NRCFs-derived EV induce autophagy in NRCFs perhaps partly through the activation of AMPKα.

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

Extracellular vesicles (EV) consist of a lipid-bilayered membrane and are typically classified as small EV (sEV or exosome) or large EV (or microvesicle). sEV mediate cell-to-cell communication and play a key role in various disease states. We recently reported that plasma sEV in normotensive Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), an animal model of human essential hypertension, regulate systemic blood pressure (BP). An abnormal vascular reactivity is involved in the onset and progression of hypertension. In the present study, we tested the hypothesis that plasma sEV may affect the reactivity of isolated blood vessels. sEV were isolated from plasma in male WKY and SHR (WsEV and SsEV, respectively) by precipitation with polyethylene-glycol and ultracentrifugation. The particle distribution and concentration of sEV were measured by a tunable resistive pulse sensing method. Isolated mesenteric arteries from normal male Wistar rats were cultured for 24 hr with WsEV, SsEV, or vehicle. There was no difference in particle distribution and total concentration between WsEV and SsEV. Both SsEV and WsEV had no significant effect on the KCl-induced maximal contraction, while SsEV specifically attenuated contraction induced by noradrenaline compared with WsEV- and vehicle-treatment. In summary, it was for the first time revealed that SsEV attenuate the agonist-induced contractility of isolated blood vessels, which might be at least partly responsible for the BP regulation by SsEV.

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

Small extracellular vesicles (sEV) contain various molecules and mediate cell-to-cell communication under both physiological and pathological conditions. We have recently reported that sEV isolated from plasma of normotensive Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) regulate systemic blood pressure. The initiation and development of hypertension partly rely on proliferation and migration of vascular smooth muscle cells (SMCs) followed by the structural remodeling of vascular wall. In the present study, we examined the effects of plasma sEV in WKY and SHR on the proliferative and migratory functions of primary rat aortic SMCs. There was no difference in the concentration and size distribution of plasma sEV between WKY and SHR, while the protein expression of CD81 in plasma sEV from SHR was lower than that from WKY. Both plasma sEV from WKY and SHR were internalized into SMCs and stimulated the migration and proliferation with a similar potency. In summary, we, for the first time, demonstrated that plasma sEV in WKY and SHR are physiologically active in terms of proliferative and migratory functions, however, these effects do not seem to be related to the pathogenesis of hypertension development.

Optimal Isolation Method of Small Extracellular Vesicles from Rat Plasma.

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.