ECTS Abstracts (2015) 1 P145

Extracellular vesicles (EVs) as shuttles of bioactive molecules: an efficient way of osteoblasts to smartly deliver Receptor Activator of Nuclear factor kappa-B Ligand (RANKL)

Alfredo Cappariello1,2, Riccardo Paone2, Mattia Capulli2, Maurizio Muraca1 & Anna Teti2

1Children Hospital Bambino Gesù, Rome, Italy; 2University of L’Aquila, L’Aquila, Italy.

Bone is the site of crowded cell-to-cell crosstalk, and various molecules are exchanged to ensure tissue homeostasis. A new mechanism of intercellular communication is represented by EVs, complex biphospholipidic structures, sized 50-1000 nm. EVs shuttle bioactive molecules, including mRNAs, miRNAs and proteins, to target cells and are involved both in physiological and pathological processes. To investigate the EV-shuttled communication between bone cells, we isolated EV pellets from osteoblast conditioned media (3.03±0.79mg), increasing their yield by 10−8M hrPTH(1-34) (4.05±1.19mg,p=0.0405). By FACS we sorted 16.67±1.93% events showing, by transmission electron microscopy, membrane integrity, and size and structure typical of EVs. EVs shuttled fluorochromes into osteoblasts, monocytes and endothelial cells. 97.1+0.26% EVs contained RNAs transferred to target osteoblasts. 53.95±3.48% EVs were RANKL-positive, which increased up to 63.6±4.20% after PTH treatment (p=0.037). EVs targeted the bone tissue ex-vivo, because murine calvaria, incubated with fluorochrome-loaded EVs, showed fluorochrome integration in bone cells with a vesicular pattern. We injected i.p. 30.000 FACS-sorted RANKL-positive EVs in 5 days-old CD1 pups and observed a fast uptake of EV-shuttled fluorochrome in bone, peaking at 1.5 hours from injection and declining thereafter to a lower plateau within 24 hours. To investigate the in-vivo impact of RANKL-positive EVs on osteoclastogenesis, we injected i.p. 4 days-old RANKL-/- mice with 30,000, 60,000, and 120,000 RANKL-positive EVs/mouse, every other day for 5 times. Tibia sections revealed Tartrate-Resistant Acid Phosphatase (TRAcP) positive cells in treated mice, which were instead totally absent in vehicle-treated RANKL-/- mice. TRAcP-positive cell area steadily increased with increasing EV densities (PBS: ND; 30,000 EVs: 398.92+54.97 μm2; 60,000 EVs: 810.17+169 μm2; 120,000 EVs: 2403.91+932.30 μm2, p<0.05), indicating dose-dependent osteoclastogenic potential. Our data demonstrate that EVs are physiologically involved in intercellular communication in bone an contribute to RANKL-induced osteoclastogenesis, representing a potential means of targeted therapeutic delivery.

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