Spread of senescence and joint inflammation via connexin43-positive exosomes released by osteoarthritic chondrocytes

Abstract

Background: Chondrocytes (CHs) in articular cartilage undergo phenotypic changes and senescence, restricting cartilage regeneration and favoring osteoarthritis (OA) progression. CHs and synovial cells from OA patients show a chronic increase in the channel protein connexin43 (Cx43), which regulates signal transduction. Extracellular vesicles (EVs), including exosomes, have been shown to play important roles in many biological functions and harbour Cx channels that allow the formation of gap junctions (GJs) between the exosome and the target cell, but the role of these EVs and exosomal-Cx43 in OA progression has not been studied yet.

Objectives: The objective of this study was to investigate the role of EVs released by OA chondrocytes (OACs) in cellular plasticity, inflammation and senescence of surrounding joint tissues.

Methods: CHs, bone and synovial cells were isolated from healthy and OA donors. EVs were obtained by ultracentrifugation and their protein content was analysed by LC-MS/MS. Protein levels were evaluated by western blot, immunofluorescence and flow cytometry. RNA expression was evaluated by RT-qPCR. Senescence and GJ intercellular communication was studied by flow cytometry and scrape loading assay, respectively.

Results: OACs showed increased levels of Cx43 within their EVs in comparison to the EVs isolated from healthy donors. Overexpression of Cx43 in CHs increased senescence and exosomal Cx43 levels. Interestingly, the treatment of CHs, bone cells and synoviocytes (target cells) with Cx43-EVs 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 to a less differentiated state via EMT by activation of Twist-1, associated with increased levels of the mesenchymal markers, as CD105/CD166. The phenotypic changes detected in OACs lead to a decrease in Col2A1 and aggrecan expression in CHs, and increased the levels of cellular senescence and the senescence-associated secretory phenotype (SASP) in the target cells 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, finding enrichment in proteins related with catabolic, senescence and wound-healing pathways.

Conclusion: Our results indicate that Cx43-positive exosomes released by OACs may be involved in the spread of cellular senescence and inflammation involved in wound healing failure. Further understanding of the role of exosomal Cx43 in OA will help to halt the disease spread and progression.