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.

Purpose: Osteoarthritis (OA) is no longer considered to be just a disease of the cartilage but has aspects of low grade inflammation and disruption of synovial homeostasis. Recent insights suggest that 70% of OA patients present with synovial inflammation (synovitis), which significantly correlates with pain and cartilage damage. Disruption of joint tissue homeostasis, results in an altered secretory profile from the synovial tissue, with microvesicles and 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 proteins, lipids and various types of RNA. Protease activated receptor 2 (PAR2) is expressed on OA synovial tissue and cartilage and is known to drive inflammation. Additionally, it has been shown to be a critical receptor in OA pathophysiology, as its knockdown in murine models results in protection from cartilage erosion. The aim of this study was to characterise the PAR2-driven secretome of OA synovial tissue, including the exosome content. The impact of isolated OA synovial exosomes on catabolic gene expression in primary OA chondrocytes was subsequently evaluated. Methods: Human OA synovial explants (n=35 collected by arthroplasty) were cultured in the presence/absence of IL-1β, PAR2 agonist peptide (SLIGKV-NH2) or a reverse peptide (RP) control. Conditioned medium (CM) was harvested after 48h and used for exosome preparation. Dead cells and debris were eliminated by differential centrifugation, with exosomes isolated by ultracentrifuge at 100,000g. Western blot analysis was used to evaluate the presence of exosome markers (CD9, CD81, HSP70 and CD63), with scanning electron microscopy (SEM) used to verify exosome presence and size between 30-150 nm. Nanoparticle Tracking Analysis (NTA) was also undertaken on exosome preparations to confirm concentration and size. Isolated exosomes were labelled with Exo-Red (staining RNA) or Exo-Green (protein) and uptake by primary human OA articular chondrocytes (n=6) evaluated using immunofluorescence imaging. Gene expression changes post exosome uptake by chondrocytes (n=4) was analysed with real time qPCR. Cytokine and MMP levels in CM were evaluated using ELISA. Results: Exosome preparations were evaluated and confirmed presence of exosome-associated markers CD9, CD81, HSP70 and CD63. NTA and SEM data confirmed that within preparations, 80-90% of microvesicles were of exosome size, ranging from 30 to 150 nm. Stimulation with IL-1β or SLIGKV-NH2 did not significantly alter protein concentration. Exosomes carried both protein and RNA cargo and were taken up by primary chondrocytes after 4h exposure. Primary chondrocytes that had taken up IL-1β stimulated synovial membrane derived exosomes, showed significant decreases in COL2A1 (p<0.001) and ACAN (p<0.001) expression. Exosomes derived from stimulation with SLIGKV-NH2 showed similar patterns, with a significant decrease in COL2A1 (p<0.01), ACAN (p<0.01) and SOX9 (p<0.01) expression. Levels of IL-6, TNFα and MMP-3 in CM were significantly increased by both IL-1β and SLIGKV-NH2 (p<0.001) stimulated samples compared to controls. Interestingly, only SLIGKV-NH2 significantly increased IL-8 (p<0.001), demonstrating differential regulation by PAR2. Conclusions: OA synovial tissue has the potential to impact chondrocyte behaviour through the release of a secretome, which includes potentially regulatory exosomes. Future characterisation of exosome cargo, will provide new insight into the role of the synovium in OA cartilage pathology.

SUMMARY Techniques for pre‐operative localization of aldosterone‐secreting adrenal adenomas were studied in thirty‐seven patients, each with hypertension and biochemical evidence of primary hyperaldosteronism and each later having adrenal surgery (thirty‐two adenomas, five bilateral adrenal hyperplasia). Bilateral adrenal vein catheterization was attempted in all cases; it was successful on the left side in all patients and in 92% of cases on the right. Adrenal vein plasma samples were obtained from the left side in 92% and from the right in 73% of cases. Adrenal vein plasma aldosterone measurements correctly indicated the presence of tumour in twenty‐eight cases but falsely predicted unilateral adenoma in two cases of bilateral adrenal hyperplasia. Adrenal venography also correctly predicted unilateral adrenal adenomas in twenty‐six cases but falsely suggested the presence of tumour in three cases of bilateral adrenal hyperplasia. Computed tomography (CT) was used in the last eight cases. In seven instances the predictions (six adenomas, one bilateral adrenal hyperplasia) were confirmed at surgery. However, the remaining patient harboured an adenoma 20 mm in diameter which was not detected by CT although diagnosed both by adrenal venography and adrenal vein aldosterone measurements. Ultrasound detected adenoma in only three of twenty‐two cases examined. Although further comparative studies of the type described here are required, the results of computed tomography are promising and suggest that this non‐invasive technique might well become the first choice procedure in localizing aldosterone‐secreting adenomas.