A. Otahal (Krems, AT)
Danube University Krems Regenerative MedicinePresenter Of 1 Presentation
P202 - Association of Regenerative Capacity of Blood EVs on Primary Osteoarthritic Chondrocytes with EV-associated miRNAs
Abstract
Purpose
Regenerative approaches in osteoarthritis therapy increasingly focus on blood products. Frequently, citrate-anti-coagulated platelet-rich plasma (CPRP) is intra-articularly injected into diseased joints, however, cell-free alternatives such as hyperacute serum (hypACT) are under development. The discovery of extracellular vesicles (EVs) in blood and EV-associated cargo molecules such as miRNAs opened up new levels of complexity in understanding the mechanisms of action and the therapeutic potential of blood products.
Methods and Materials
To investigate the roles of EVs isolated from CPRP and hypACT during osteoarthritis (OA), primary human OA chondrocytes were treated with EVs enriched via ultracentrifugation (UC) from these blood products. Chondroprotective and anti-inflammatory effects were evaluated based on gene expression analysis via RT-qPCR and Western Blot, and cytokine release via ELISA. EV-associated miRNA profiles were analysed by screening a 372 miRNA panel via RT-qPCR in EVs purified via UC and size exclusion chromatography (SEC).
Results
EVs increased the expression of anabolic markers type II collagen (COL2A1), SRY-box transcription factor 9 (SOX9) and aggrecan (ACAN), but also the catabolic marker matrix metalloproteinase 3 (MMP3). CPRP blood product increased SOX9 protein expression; in contrast, CPRP EVs decreased NFκB and COX2 expression in IL1β-stimulated chondrocytes compared to unstimulated cells. hypACT EVs induced SOX9 protein expression while preventing IL6 secretion compared to hypACT blood product. Analysis of the functional repertoire encoded in EV-associated miRNAs revealed that CPRP EV-associated miRNAs target NFκB signaling and hypACT EV-associated miRNAs affect IL6 and TGFβ/SMAD signaling.
Conclusion
Blood EVs are sufficient to increase chondrotypic gene expression in OA chondrocytes, while preventing pro-inflammatory cytokine release compared to full blood products. The effects observed in the biological assays correlate with the functional repertoire of EV-associated miRNAs. This highlights the potential of blood EVs and their cargo to be regulators of cartilage matrix metabolism and inflammation as well as candidates for new cell-free therapeutic approaches for OA.
Presenter Of 1 Presentation
P202 - Association of Regenerative Capacity of Blood EVs on Primary Osteoarthritic Chondrocytes with EV-associated miRNAs
Abstract
Purpose
Regenerative approaches in osteoarthritis therapy increasingly focus on blood products. Frequently, citrate-anti-coagulated platelet-rich plasma (CPRP) is intra-articularly injected into diseased joints, however, cell-free alternatives such as hyperacute serum (hypACT) are under development. The discovery of extracellular vesicles (EVs) in blood and EV-associated cargo molecules such as miRNAs opened up new levels of complexity in understanding the mechanisms of action and the therapeutic potential of blood products.
Methods and Materials
To investigate the roles of EVs isolated from CPRP and hypACT during osteoarthritis (OA), primary human OA chondrocytes were treated with EVs enriched via ultracentrifugation (UC) from these blood products. Chondroprotective and anti-inflammatory effects were evaluated based on gene expression analysis via RT-qPCR and Western Blot, and cytokine release via ELISA. EV-associated miRNA profiles were analysed by screening a 372 miRNA panel via RT-qPCR in EVs purified via UC and size exclusion chromatography (SEC).
Results
EVs increased the expression of anabolic markers type II collagen (COL2A1), SRY-box transcription factor 9 (SOX9) and aggrecan (ACAN), but also the catabolic marker matrix metalloproteinase 3 (MMP3). CPRP blood product increased SOX9 protein expression; in contrast, CPRP EVs decreased NFκB and COX2 expression in IL1β-stimulated chondrocytes compared to unstimulated cells. hypACT EVs induced SOX9 protein expression while preventing IL6 secretion compared to hypACT blood product. Analysis of the functional repertoire encoded in EV-associated miRNAs revealed that CPRP EV-associated miRNAs target NFκB signaling and hypACT EV-associated miRNAs affect IL6 and TGFβ/SMAD signaling.
Conclusion
Blood EVs are sufficient to increase chondrotypic gene expression in OA chondrocytes, while preventing pro-inflammatory cytokine release compared to full blood products. The effects observed in the biological assays correlate with the functional repertoire of EV-associated miRNAs. This highlights the potential of blood EVs and their cargo to be regulators of cartilage matrix metabolism and inflammation as well as candidates for new cell-free therapeutic approaches for OA.