0081: Trans-endothelial Trafficking of Fibroblast-like Synoviocytes Amplifies Synovial Inflammation in Rheumatoid Arthritis

Background/Purpose: The discovery of circulating fibroblast-like synoviocyte (FLS), known as PRIME cells, in RA patients suggests that synovial FLS may enter the circulation through blood vessels and contribute to systemic inflammation. This study aimed to detect trans-endothelial migration of FLS and dissect the molecular crosstalk between FLS and endothelial cells (EC) during this process and its relevance in rheumatoid inflammation.

Methods: Human and mouse arthritic synovium were analyzed using immunofluorescence and RNA in situ hybridization (ISH) to identify trans-endothelial migrating FLS. An in vitro trans-endothelial migration model was established using RA FLS and human umbilical vein ECs (HUVEC), followed by transcriptomics and proteomic analysis. Spatial transcriptomics was performed on RA synovial biopsies. Functional experiments were conducted through gene knockdown in vitro and in vivo.

Results: We identified FLS transmigration through EC layers of capillaries in RA synovium, but not in osteoarthritis (OA), which correlated with RA disease activity. ISH confirmed the presence of intravascular FLS in human and mouse arthritic synovium (Fig. 1A, B, C, R²=0.6, P=0.0004). In vitro trans-endothelial migration assay demonstrated that RA FLS actively transmigrate across EC layers. Bulk RNA-seq of trans-endothelial migrated FLS identified increased expression of polycystin-1 (PKD1) (Fig. 2A). Integration of transcriptomics and proteomic data identified cathepsin K (CTSK) as upregulated in EC after transmigration of FLS (Fig 2B), Spatial transcriptomics studies showed that PKD1⁺ FLS are located in close proximity to CTSK⁺ ECs in RA synovium, supporting a spatially organized induction of CTSK by FLS in EC. In vitro assays confirmed that PKD1 knockdown significantly impaired FLS transmigration across EC layers while PKD1 knockdown in FLS reduced the severity of arthritis in vivo (Fig. 3A, B). CTSK knockdown in ECs exposed to FLS impaired angiogenesis while systemic administration of the CTSK inhibitor Odanacatib attenuated joint inflammation and vascular leakage in arthritic mice.

Conclusion: Our findings demonstrate that FLS undergo trans-endothelial migration in RA synovium and identify a mechanism driven by such migration and involving PKD1 and CTSK, which amplifies EC-driven inflammation and RA severity. These results might also provide mechanistic insight into the generation of PRIME cells and highlight FLS–EC spatial interactions as a potential therapeutic target in RA.