Temporal extracellular vesicle protein changes following intraarticular treatment with integrin α10β1-selected mesenchymal stem cells in equine osteoarthritis

Abstract

IntroductionEquine osteoarthritis (OA) is a heterogeneous, degenerative disease of the musculoskeletal system with multifactorial causation, characterized by a joint metabolic imbalance. Extracellular vesicles are nanoparticles involved in intracellular communication. Mesenchymal stem cell (MSC) therapy is a form of regenerative medicine that utilizes their properties to repair damaged tissues. Despite its wide use in veterinary practice, the exact mechanism of action of MSCs is not fully understood. The aim of this study was to determine the synovial fluid extracellular vesicle protein cargo following integrin α10β1-selected mesenchymal stem cell (integrin α10-MSC) treatment in an experimental model of equine osteoarthritis with longitudinal sampling.MethodsAdipose tissue derived, integrin α10-MSCs were injected intraarticularly in six horses 18 days after experimental induction of OA. Synovial fluid samples were collected at day 0, 18, 21, 28, 35, and 70. Synovial fluid was processed and extracellular vesicles were isolated and characterized. Extracellular vesicle cargo was then analyzed using data independent acquisition mass spectrometry proteomics.ResultsA total of 442 proteins were identified across all samples, with 48 proteins differentially expressed (FDR ≤ 0.05) between sham-operated control joint without MSC treatment and OA joint treated with MSCs. The most significant pathways following functional enrichment analysis of the differentially abundant protein dataset were serine endopeptidase activity (p = 0.023), complement activation (classical pathway) (p = 0.023), and collagen containing extracellular matrix (p = 0.034). Due to the lack of an OA group without MSC treatment, findings cannot be directly correlated to only MSCs.DiscussionTo date this is the first study to quantify the global extracellular vesicle proteome in synovial fluid following MSC treatment of osteoarthritis. Changes in the proteome of the synovial fluid-derived EVs following MSC injection suggest EVs may play a role in mediating the effect of cell therapy through altered joint homeostasis. This is an important step toward understanding the potential therapeutic mechanisms of MSC therapy, ultimately enabling the improvement of therapeutic efficacy.

Multi-Omic Temporal Landscape of Plasma and Synovial Fluid-Derived Extracellular Vesicles Using an Experimental Model of Equine Osteoarthritis

“…ORA analysis identified several terms that were functionally enriched in EV cargo for both biofluids compared to the whole proteome including 'complement activation classical pathway', 'complement activation alternative pathway' and 'serine-type endopeptidase inhibitor activity'. These terms are all in keeping with the known role of EVs driving tissue inflammation and repair [15,[41][42][43][44] and support their potential as biomarkers of OA progression. However, there were also terms unique to each biofluid.…”

“…For synovial fluid-derived EVs, 11 mL equine synovial fluid was used. This was a SF pool derived from our equine musculoskeletal biobank (VREC561), with samples collected from our Clarke et al study [15] and samples from the present study. For both libraries, the pools were centrifuged at 100,000× g and 4 • C for 70 min and then resuspended in 200 µL 6M urea/1M ammonium bicarbonate/0.5% sodium deoxycholate.…”

“…EV cargo is involved in cross-talk between cells within joint tissues and affects ECM turnover and inflammation [13,14], thus representing a crucial step in the regulation of OA (Supplementary File S1). The role of EVs in OA provides a foundation to create novel disease-modifying treatments [13,15]. Promising results were obtained in the therapeutic application of mesenchymal stem cell-derived EVs for cartilage repair in experimental OA [16].…”

“…Synovial fluid-derived EVs have a close relationship with the pathogenesis of arthritis [17]. Recent studies described synovial fluid-derived EVs as biomarkers for different stages of joint disease [18], whilst we [15,19,20] and others [21] have interrogated EV cargo using sequencing and metabolomics [22]. There are few studies on the EV protein cargo in synovial fluid-derived EVs in OA [23,24] and none in the horse.…”

Proteome and phospholipidome interrelationship of synovial fluid-derived extracellular vesicles in equine osteoarthritis: An exploratory ‘multi-omics’ study to identify composite biomarkers

“…Equine SF was pooled using samples from the metacarpophalangeal joint from our equine musculoskeletal biobank (VREC561) and samples collected in previous studies from the carpal and metacarpal joint of healthy horses as well as those with OA, resulting in a total of 11 ml pooled SF (n = 1) for library generation. These samples were analysed as previously described in order to generate the necessary reference library [ 38 ].…”