Oru (INIBIC), A Coru , Spain; cProteomics laboratory. Instituto de Investigaci Sanitaria de Santiago de Compostela (IDIS), Complexo CD33 Proteins Accession Hospitalario Universitario de Santiago de Compostela (CHUS), A Coru , Spain; dUnit of Experimental Neurology-Neurobiology., Madrid, Spain; eBone and Joint Research Unit, Rheumatology Division, IIS-Fundaci Jim ez D z UAM, Madrid, Spain; fDepartment of Pathology, College of Medicine, Wayne State University, Detroit, MI, USA; gTranslational Molecular Pathology investigation group. Vall d’Hebron Research Institute (VHIR), Universitat Aut oma de Barcelona, Barcelona, Spain; hDepartamento de Bioqu ica y Biolog Molecular, Instituto de Neurociencias de Castilla y Le (INCYL), Universidad de Salamanca, Salamanca, Spain; iFlow Cytometry Core Technologies, UCD Conway Institute, University College Dublin, Dublin, Ireland; jDepartment of Orthopaedic Surgery and Galanin Proteins Purity & Documentation Traumatology, Complexo Hospitalario Universitario de Santiago de Compostela (CHUS). Universidade de Santiago de Compostela (USC), Santiago de Compostela, SpainIntroduction: Chondrocytes in articular cartilage undergo phenotypic alterations and senescence, restricting cartilage regeneration and favouring osteoarthritis (OA) progression. Like other wound healing problems, chondrocytes from OA sufferers show a chronic improve inside the transmembrane channel protein connexin43 (Cx43). Extracellular vesicles (EVs), which includes exosomes, have already been show to harbour connexinJOURNAL OF EXTRACELLULAR VESICLESchannels that let the formation of gap junctions between the exosome plus the target cell. Nevertheless, the part of these vesicles and exosomal-Cx43 in OA progression has not been studied yet. The objective of this study was to investigate the role of EVs released by osteoarthritic chondrocytes (OACs) in cellular plasticity and senescence of surrounding tissues. Solutions: EVs were isolated from OA/healthy chondrocytes by ultracentrifugation and their protein content was analysed by LC-MS/MS applying 6600 triple TOF. RNA levels, protein activity and cellular senescence were analysed by RT-qPCR, western blot, immunofluorescence and flow cytometry. Outcomes: Our results indicate that OACs contain enhanced levels of Cx43 inside their EVs in comparison to the EVs isolated from wholesome donors. Overexpression of Cx43 in chondrocytes improved senescence plus the total content material of Cx43 inside the EVs. The remedy of target cells with EVs containing Cx43 led to a important improve in Cx43 mRNA and protein levels. The increase of Cx43 cause dedifferentiation inside the recipient cells via EMT by activation of Twist-1, with increased levels on the mesenchymal markers CD105 and CD166. The phenotypic changes detected in OACs bring about a reduce within the key cartilage markers Col2A1 and ACAN expression, and enhanced the levels of cellular senescence and SASP in target cells via p53/p16 and NF-k These final results had been corroborated by analysing the protein cargo of those Cx43 constructive EVs, exactly where we discovered enrichment in proteins connected with the catabolic, senescence and wound-healing pathways Summary/Conclusion: With each other, these benefits recommend that Cx43-positive EVs released by OACs could be involved in the spread of cellular senescence, inflammation and reprogramming elements involved in wound healing failure to neighbouring tissues in the joint. Further understanding in the role of exosomal Cx43 in OA will assistance to halt the disease spread and progression.OF17.Extracellular vesicles in ageing: from skin to bone.