High-resolution flow cytometric analysis of synovial fluid derived extracellular vesicle populations during joint inflammation

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    Introduction: Extracellular vesicles (EVs) in the synovial fluid are likely to play a role in the communication between articular cells and tissues during health and disease. Gaining insight into this form of intercellular communication may be of great benefit for the progress of cartilage regenerative medicine and treatment of joint disease. Because EVs are known to play an important role in inflammatory processes, in this study synovial fluid derived EVs from equine joints with and without LPS-induced inflammation were isolated and analyzed using high-resolution flow cytometry.
    Methods: Synovitis was induced in middle carpal joints of Warmblood horses by injection of 0.5 ng lipopolysaccharide (LPS) from E. coli into each joint. Synovial fluid samples were collected at 0h, 8h, 24h and 168h post LPS injection. Synovial fluid EVs were isolated using an optimized protocol and pelleted at 10,000g and 100,000g respectively, labeled with PKH67 and separated according to buoyant density by iodixanol gradient ultracentrifugation. Concentrations of PKH67-labeled EVs were analyzed by high-resolution flow cytometry (BD Influx). To further characterize the different EV subsets lipidomics using HPLC/LCMS is currently performed.
    Results: Using high resolution flow cytometry differences between EVs present in the 10,000g and 100,000g pellets were readily observed based on the light scattering patterns of individual EVs. Quantitative EV analysis revealed that the
    highest concentration of EVs, derived from both the 10,000g and 100,000g pellets, were found at 8h post LPS injection while concentrations gradually returned to baseline at 168h. These findings are in line with previous measurements of inflammation markers (prostaglandin E2, substance P, bradykinin, MMP activity) and leukocyte and neutrophil infiltration in the same samples, which also showed a peak at 8h post LPS injection [De Grauw et al. Arthritis Res Ther 2009;11:R35]
    Interestingly, differences in kinetics were observed between the 10,000g and 100,000g EVs, suggesting subset specific
    characteristics for production/infiltration or clearance of these vesicles in the joint after an acute inflammatory insult.
    Summary/Conclusion: These data show that EV concentrations in synovial fluid increase during inflammatory responses in the joint, suggesting a role for EV-mediated signaling in this process. To characterize the different EV-subsets
    comprehensive lipid analysis is currently performed to define specific lipid profiles for EV subpopulations. Further studies will be undertaken to examine the cellular origin of these EVs and their specific function during inflammation.
    Original languageEnglish
    Publication statusPublished - 22 Apr 2015
    EventISEV annual meeting 2015 - Bethesda North Marriott, Washington, DC, United States
    Duration: 22 Apr 201526 Apr 2015


    ConferenceISEV annual meeting 2015
    Country/TerritoryUnited States
    CityWashington, DC


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