Differential effects of exosomes derived from separate osteoarthritis synovial compartments: implications for erosive cartilage disease

S. Asghar, G. Litherland, D. Meek, J. Cole, J. Lockhart, C.S. Goodyear, A. Crilly

Research output: Contribution to journalMeeting Abstract

Abstract

Purpose: Osteoarthritis (OA) is no longer considered to be just a disease of the cartilage but often exhibits low-grade inflammation and disruption of synovial tissue homeostasis. Recent work would suggest that 70% of OA patients present with synovial inflammation, which significantly correlates with pain and cartilage damage. Disruption of joint tissue homeostasis results in an altered secretory profile from the synovial membrane, with extracellular vesicles such as exosomes contributing to this secretome. Importantly, exosomes have been implicated in cell communication through their ability to carry and transfer a range of potentially modulatory/regulatory cargo, including microRNAs. Protease activated receptor 2 (PAR2) has been identified as a critical regulatory molecule for inflammatory joint disease and OA pathophysiology. The aim of this study was to characterise and compare the PAR2 driven secretome of OA tissues and cells, including the exosome content.
Methods: Equal weights of human OA synovial membrane and infrapatellar white adipose tissue (WAT) explants (n=50 collected by arthroplasty) were cultured in the presence/absence of IL-1β, PAR2 peptide agonist (SLIGKV-NH2) or a reverse peptide (RP) control and conditioned media harvested after 48h and used for exosome preparation. Parallel experiments were run with synovial fibroblasts. Dead cells and debris were eliminated by differential centrifugation, with exosomes isolated by ultracentrifuge at 100,000g. Exosomes were characterised by western blot, scanning electron microscopy (SEM) and Nanoparticle Tracking Analysis (NTA). Following characterisation, exosomes were labelled with Exo-Red (staining RNA) or Exo-Green (protein) and uptake by primary OA articular chondrocytes (n=6) evaluated using immunofluorescence imaging. Gene expression and cytokine changes 6h post exosome uptake by chondrocytes (n=9) was analysed with real time qPCR and ELISA. RNA was isolated from non-stimulated, IL-1β, SLIGKV-NH2 and RP derived synovial membrane exosomes as well as non-stimulated/IL-1β synovial fibroblast derived exosomes and their parental cells. Once enriched for small RNA, libraries were prepared, sequenced and bioinformatics analysis undertaken.
Results: Exosome preparations from synovial fibroblasts, synovial membrane and WAT explants were evaluated and presence of exosome-associated markers CD9, CD81, HSP70 and CD63 confirmed. Furthermore, NTA and SEM demonstrated that within preparations, 80-90% of microvesicles were of exosome size; ranging from 30 to 150nm. Exosomes carried both protein and RNA cargo and were taken up by 90% of primary chondrocytes after 4h exposure, regardless of source. Uptake of non-stimulated synovial membrane and WAT derived exosomes increased IL-6 (p<0.001, p<0.01 respectively) and IL-8 (p<0.05, p>0.01 respectively) protein secretion. Only synovial-derived exosomes increased MMP-3 (p<0.001) and TNFα (p<0.001) protein secretion. Regardless of source, exosomes had no significant effect on IL-10 (p<0.1) protein release from primary chondrocytes. Interestingly, exosomes derived from IL-1β stimulated WAT increased MMP-3 and TNFα (p<0.01, p<0.01) protein secretion compared to non-stimulated controls. Primary chondrocytes that had taken up exosomes (regardless of source) also showed significant transcript expression decreases in COL2A1 (p<0.001) and ACAN (p<0.001) and increased expression of MMP1 (p<0.01).
Conclusions: These findings suggest that tissues within the synovial compartment have the potential to impact cartilage destruction through exosomes release, by increasing the release of inflammatory cytokine mediators from chondrocytes and regulating expression of catabolic and anabolic genes. Further analysis of small RNA cargo of exosomes derived from different sources could give insight into novel regulatory mechanisms within the OA joint.
Original languageEnglish
Article number260
Pages (from-to)S192
Number of pages1
JournalOsteoarthritis and Cartilage
Volume27
Issue numberS1
Early online date15 Apr 2019
DOIs
Publication statusPublished - 30 Apr 2019

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