Abstract
The study of the miRNA cargo embedded in extracellular vesicles (EVs) released from adipose-derived mesenchymal stromal cells (ASC) preconditioned with IL-1β, an inflammatory stimulus driving osteoarthritis (OA), along with EVs-cartilage dynamic interaction represent poorly explored fields and are the purpose of the present research. ASCs were isolated from subcutaneous adipose tissue and EVs collected by ultracentrifugation. Shuttled miRNAs were scored by high-throughput screening and analyzed through bioinformatics approach that predicted the potentially modulated OA-related pathways. Fluorescently labeled EVs incorporation into OA cartilage explants was followed in vitro by time-lapse coherent anti-Stokes Raman scattering; second harmonic generation and two-photon excited fluorescence. After IL-1β preconditioning, 7 miRNA were up-regulated, 4 down-regulated, 37 activated and 17 silenced. Bioinformatics allowed to identify miRNAs and target genes mainly involved in Wnt, Notch, TGFβ and Indian hedgehog (IHH) pathways, cartilage homeostasis, immune/inflammatory responses, cell senescence and autophagy. As well, ASC-EVs steadily diffuse in cartilage cells and matrix, reaching a plateau 16 h after administration. Overall, ASCs preconditioned with IL-1β allows secretion of EVs embedded with a chondro-protective miRNA cargo, able to fast penetrate in collagen-rich areas of cartilage with tissue saturation in a day. Further functional studies exploring the EVs dose-effects are needed to achieve clinical relevance.
Highlights
Osteoarthritis (OA) is a chronic pathology of the whole joint [1], leading to a progressive loss of bony, cartilaginous and synovial tissues homeostasis, driven and sustained by catabolic and inflammatory process.Adipose-derived mesenchymal stromal cells (ASCs) based therapy has been used in some clinical trials to treat OA patients [2] as a tool potentially acting on several pathological targets given their immunomodulatory and trophic ability
ASCs showed high responsiveness to IL-1β stimulation, resulting in the up-regulation of matrix metalloproteases (MMPs)-1, -3 and -13, angiogenic and mitogenic factors, cytokines controlling production, differentiation and function of macrophages, as well as molecules belonging to the insulin-like growth factor (IGF), platelet-derived growth factor (PDGF) and transforming growth factor (TGF) families and pro- (IL-1β and IL-8) and anti- (IL-1Ra) inflammatory cytokines [3]
Among the IL-1β up-regulated miRNA, miR-155-5p followed by miR-146a-5p, miR196b-5p, miR-125a-3p, miR-134-5p and miR-520c-3p showed the highest number of interactions (Table S2)
Summary
Adipose-derived mesenchymal stromal cells (ASCs) based therapy has been used in some clinical trials to treat OA patients [2] as a tool potentially acting on several pathological targets given their immunomodulatory and trophic ability. ASCs respond in an adaptive way to inflammatory stimuli, in particular to interleukin-1β (IL-1β) [3], a central molecular regulator of the complex network of proteolytic enzymes, chemokines and cytokines leading the degenerative processes of OA [4]. ASCs showed high responsiveness to IL-1β stimulation, resulting in the up-regulation of matrix metalloproteases (MMPs)-1, -3 and -13, angiogenic and mitogenic factors, cytokines controlling production, differentiation and function of macrophages, as well as molecules belonging to the insulin-like growth factor (IGF), platelet-derived growth factor (PDGF) and transforming growth factor (TGF) families and pro- (IL-1β and IL-8) and anti- (IL-1Ra) inflammatory cytokines [3]. ASCs inflammatory priming with interferon γ (IFNγ) determined a production of a 34.7% chondro-protective versus a 13.3% chondro-destructive miRNAs and a secretion of 26.2% versus 4.5% of M2- versus M1-macrophages-related miRNAs [9]
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