Background/Purpose: Soluble CD13 (sCD13), the soluble form of aminopeptidase N/CD13, exhibits potent chemoattractant, angiogenic, and arthritogenic properties. While we previously identified certain G protein-coupled receptors (GPCR) including bradykinin receptor B1 as receptors for sCD13 in rheumatoid arthritis (RA), its complete receptor repertoire remains unclear. In this study, we aimed to investigate protease-activated receptor 4 (PAR4) as a novel receptor for sCD13.

Methods: Single-cell RNA sequencing datasets were reanalyzed to determine the expression of PAR4 on synovial cells in RA, which was then validated by immunofluorescence, Wester blotting, and flow cytometry. Induction of PAR4 in human umbilical vein endothelial cells (HUVECs) by TNF-a or IL-1b was assessed by qPCR and Western blotting. PAR4 was overexpressed in RA fibroblast-like synoviocytes (FLS), and cytokine production in sCD13-treated FLS was determined by qPCR and ELISA. The effect of CD13 blocking antibodies (WM15) on proliferation of FLS was dynamically analyzed using the IncuCyte. Effects of PAR4 knockdown or blocker (BMS986120) on the migration and angiogenesis abilities of HUVECs were evaluated by Transwell chambers and tube formation assay, respectively. The binding model of CD13-PAR4 was predicted and constructed using AlphaFold-multimer. Enzymatic inhibitor for CD13, bestatin, was also utilized to assess the enzymatic mechanism of sCD13 activating PAR4. Western blot was performed to identify signaling pathways stimulated by sCD13 in the presence of BMS986120 or WM15. BMS986120 was administered orally to collagen-induced arthritis (CIA) mice and joint inflammation was evaluated.

Results: PAR4 was abundantly expressed in RA synovium, especially in endothelial cells and FLS. While the expression of PAR4 in RA synovium was significantly higher compared to normal and osteoarthritis synovium, it was not induced by TNF-a or IL-1b. In RA FLS, PAR4 overexpression augmented sCD13-mediated proliferation, cytokine production, and ERK1/2 signaling. Both blocking and knocking down PAR4 could suppress sCD13-induced HUVEC migration and angiogenesis in vitro. BMS986120 also suppressed sCD13-mediated ERK1/2 phosphorylation in HUVECs. In addition, The CD13-PAR4 interaction involves the center domain of CD13 and the PAR4’s pro-peptide region, which needs to be cleaved off for receptor activation. Inhibiting enzymatic activities of sCD13 with bestatin partially reduced sCD13 stimulated migration and angiogenesis of HUVECs. In vivo, BMS986120 attenuated joint synovitis and bone erosion in CIA mice.

Conclusion: We identified PAR4 as a second functional sCD13 receptor that mediates proinflammatory signaling in RA FLS and angiogenic responses in endothelial cells, which positions PAR4 as a therapeutic target in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/soluble-cd13-engages-protease-activated-receptor-4-to-promote-synovial-inflammation-and-angiogenesis-in-rheumatoid-arthritis/