INVESTIGATING JOINT PATHOPHYSIOLOGY THROUGH SYNOVIAL FLUID PROTEOMICS, IMMUNE CELL PROFILING, AND THERAPEUTIC APPROACHES IN EQUINE OSTEOARTHRITIS
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The objective of this thesis research was to investigate pathophysiological processes, cell populations, and therapeutic mechanisms in equine osteoarthritis (OA) diagnosis and management. This was achieved through four specific aims – 1) to explore the synovial fluid proteome in equine carpal OA, 2) to identify changes in macrophage and T-cell populations along with synovial membrane (SM) transcriptomic alterations in equine fetlock OA, 3) to improve our knowledge of the effects of hydrogel and non-hydrogel intra-articular therapies on articular cartilage (AC) and SM, and 4) to explore the combination therapy of proteoglycan 4 (PRG4) and interleukin-1 receptor antagonist (IL1Ra) protein delivered using a high-capacity adenoviral gene therapy platform to mitigate the development of post-traumatic OA (PTOA). Synovial fluid (SF) from horses with carpal OA or healthy joints were subjected to liquid chromatography-tandem mass spectrometry to identify the proteins present. Differential protein analysis identified significant increases in pregnancy zone protein, alpha-2-macroglobulin, and lubricin in OA, along with a decrease in gelsolin. A larger cohort of SF samples was used to confirm the increased lubricin and decreased gelsolin using enzyme linked immunosorbent assay. SF and SM from racehorse fetlocks were used to evaluate macrophage and T-cell populations using flow cytometry in relation to osteoarthritis severity, alongside differential gene expression in SM. CD8+ T-cells, pro-inflammatory/M1 macrophages, and macrophages co-expressing M1 and M2 cell surface markers were positively correlated with OA severity. SM transcriptomics revealed several enriched pathways, including connective tissue remodeling and neuron projection organization. Equine AC and SM explants stimulated with IL-1β or TNF-α were simultaneously treated with triamcinolone acetonide, hyaluronic acid, collagen-elastin hydrogel matrix, or 2.5% polyacrylamide hydrogel. Nitric oxide and sulfated glycosaminoglycan were measured in AC media, and prostaglandin E2, CCL2, CCL3, CCL5, CCL11, TNF-α, and IL-1β in SM media. All therapeutics were successful in reducing nitric oxide production by AC and prostaglandin E2 by SM. An equine OA model was used to evaluate the efficacy of a combination proteoglycan 4 (PRG4) and interleukin 1 receptor antagonist protein therapy delivered by high-capacity adenoviral vector in reducing the severity of PTOA. Horses treated with combination therapy showed reduced lameness compared to PRG4 alone or vector control.