Connexin43-positive exosomes from osteoarthritic chondrocytes spread senescence and inflammatory mediators to nearby synovial and bone cells

Abstract

Purpose: Chondrocytes in articular cartilage undergo phenotypic changes and senescence, restricting cartilage regeneration and favoring osteoarthritis (OA) progression, characterized by degradation of articular cartilage, physical disability and pain. Like other wound healing disorders, chondrocytes and synovial cells from OA patients show a chronic increase in the transmembrane channel protein connexin43 (Cx43), which through the exchange of ions and molecule or binding of signaling factors to the membrane regulates signal transduction. Extracellular vesicles (EVs), including exosomes, have been show to play important roles in many biological functions and harbour connexin channels that allow the formation of gap junctions between the exosome and the target cell. However the role of these vesicles and exosomal-Cx43 in OA progression has not been studied yet. The main objective of this study was to investigate the role of exosomes released by osteoarthritic chondrocytes in cellular plasticity and senescence of surrounding tissues.

Methods: Chondrocytes, bone and synovial cells were isolated from human tissues from healthy and OA donors. Extracellular vesicles (EVs), including exosomes, were obtained by differential ultracentrifugation and their protein content was analysed by LC-MS/MS using 6600 triple TOF. Protein levels were evaluated by western blot, immunofluorescence and flow cytometry. RNA expression was evaluated by RT-qPCR. Gap junction intercellular communication was studied by scrape loading assay and flow cytometry.

Results: OA chondrocytes (OACs) showed increased levels of Cx43 within their EVs in comparison to the EVs isolated from healthy donors. Furthermore, overexpression of Cx43 in chondrocytes with a vector or by treatment with the mitochondrial inhibitor oligomycin increased senescence and the total content of Cx43 in the EVs. Interestingly, the treatment of chondrocytes, bone cells and synoviocytes (target cells) with EVs containing Cx43, released by OACs, led to a significant increase in both Cx43 mRNA and protein levels in the recipient cells. The increase of Cx43 in target cells acted as a positive regulator of the reversion of the recipient cells to a less differentiated state via EMT by activation of Twist-1, associated with increased levels of the mesenchymal markers CD105 and CD166, as well as N-cadherin, Slug, Vimentin and Smad2/3. The phenotypic changes detected in OACs lead to a decrease in collagen II and aggrecan expression in chondrocytes, and increased the levels of cellular senescence and the senescence-associated secretory phenotype (SASP) in target cells via p53/p16 and NF-kß. These results were corroborated by analysing the protein cargo of these Cx43 positive EVs by LC-MS/MS, where we found an enrichment in proteins related with the catabolic, senescence and wound-healing pathways together with factors of the complement system and innate immunity.

Conclusions: Our results indicate that Cx43-positive exosomes and extracellular vesicles released by osteoarthritic chondrocytes may be involved in the spread of cellular senescence and inflammatory and reprogramming factors involved in wound healing failure to neighbouring tissues in the joint, spreading OA among cartilage, synovium and subchondral bone. The results suggest that further understanding of the role of exosomal Cx43 in OA will help to halt the disease spread and progression.