Abstract
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, synovial fluid derived EVs from equine joints with and without LPS-induced inflammation were isolated and analyzed using quantitative and qualitative techniques.
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, 100,000g and 200,000g consecutively, labeled with PKH67 and separated according to buoyant density by iodixanol gradient ultracentrifugation. Isolated EVs were visualized by cryo-electron microscopy, concentrations of PKH67-labeled EVs were determined by high-resolution flow cytometry (BD Influx) and lipids were analyzed by HPLC/LCMS.
Results - During acute joint inflammation highest concentrations of EVs were found at 8h and 24h post LPS injection. At 168h EV concentrations returned to baseline. This is in line with inflammation markers and leukocyte/neutrophil infiltration in fresh synovial fluid samples from the same study. Cryo-electron microscopy verified the presence of EVs, which were heterogeneous in size and shape. Specific EV-associated lipids were increasingly present in the synovial fluid of horses with acute joint inflammation compared to healthy controls.
Conclusion - EV concentrations in synovial fluid increase during inflammatory responses in the joint and EV lipid composition changes after a single inflammatory insult, suggesting EV-mediated lipid transport and signaling during acute synovitis. Ongoing functional assays will verify the role of EVs and their associated lipids in intracellular communication during synovitis.
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, 100,000g and 200,000g consecutively, labeled with PKH67 and separated according to buoyant density by iodixanol gradient ultracentrifugation. Isolated EVs were visualized by cryo-electron microscopy, concentrations of PKH67-labeled EVs were determined by high-resolution flow cytometry (BD Influx) and lipids were analyzed by HPLC/LCMS.
Results - During acute joint inflammation highest concentrations of EVs were found at 8h and 24h post LPS injection. At 168h EV concentrations returned to baseline. This is in line with inflammation markers and leukocyte/neutrophil infiltration in fresh synovial fluid samples from the same study. Cryo-electron microscopy verified the presence of EVs, which were heterogeneous in size and shape. Specific EV-associated lipids were increasingly present in the synovial fluid of horses with acute joint inflammation compared to healthy controls.
Conclusion - EV concentrations in synovial fluid increase during inflammatory responses in the joint and EV lipid composition changes after a single inflammatory insult, suggesting EV-mediated lipid transport and signaling during acute synovitis. Ongoing functional assays will verify the role of EVs and their associated lipids in intracellular communication during synovitis.
| Original language | English |
|---|---|
| Publication status | Unpublished - 19 Nov 2015 |
| Event | Veterinary Science Day - Bunnik, Netherlands Duration: 19 Nov 2015 → … |
Conference
| Conference | Veterinary Science Day |
|---|---|
| Country/Territory | Netherlands |
| City | Bunnik |
| Period | 19/11/15 → … |