Aminopeptidase N/CD13, is a transmembrane, cytokine-inducible ectopeptidase, highly expressed by rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), monocytes (MNs), and endothelial cells (ECs). CD13 is shed from the FLS surface and high levels of soluble (s)CD13 are found in RA synovial fluid (SF). sCD13 is a potent chemoattractant for activated T cells, MNs, and ECs, and depletion of sCD13 from RA SF decreased T cell and MN migration. We have recently published that sCD13 is also a potent angiogenic factor and mediates its actions through engagement of a G-protein-coupled receptor (GPCR). We have identified the receptor for sCD13 as the bradykinin B1 receptor (B1R), a GPCR. A B1R antagonist inhibited K/BxN serum transfer arthritis, zymosan induced arthritis in mice, MN migration, and phosphorylation of signaling molecules in RA FLS. We have found, using an enzymatically inactive mutant, that most of the effects of sCD13, are independent of its enzymatic activity. Moreover, Cd13 knockout mice are resistant to acute arthritis development when injected with tumor necrosis factor-?. This proposal will determine the roles of CD13 and B1R in RA angiogenesis, MN ingress, and murine models of arthritis. The role of sCD13 and B1R in acute inflammatory arthritis will be evaluated by injecting pro-inflammatory factors into knees of Cd13-/-, B1R-/-, and wild type (wt) mice. The RA model, collagen induced arthritis (CIA), will be performed in Cd13-/-, B1R-/-, and DBA/1J wt mice. We will compare arthritis severity, bone destruction, leukocyte recruitment, angiogenesis, and production of cytokines in these groups of mice. To further examine the roles of CD13 and B1R in angiogenesis, the dermal punch wound, and sponge granuloma models will be studied in Cd13-/-, B1R-/-, and wt mice. These models represent various aspects of the angiogenic processes involved in RA pathogenesis. B1R antagonists will be used to determine the role of B1R in sCD13-mediated angiogenesis, MN migration, and phosphorylation of signaling molecules. Subsets of RA FLS will be sorted by flow cytometry and the role of sCD13 and its receptor will be assessed in these FLS subpopulations. The contribution of sCD13 in RA angiogenesis will be examined by performing EC tube formation and Matrigel plug assays with CD13-depleted or sham depleted RA SFs. Sponge granuloma assays will be performed using sCD13 as a stimulus to evaluate whether defective angiogenesis can be restored in Cd13-/- mice. We will perform these assays using mutant and WT CD13 +/- B1R antagonists to demonstrate whether its enzymatic activity contributes to sCD13-mediated functions. To assess the interaction between sCD13 and B1R, an RA synovial tissue-severe combined immunodeficient mouse chimera will be performed using sCD13 +/- B1R antagonists. After accomplishing these studies, we will be able to demonstrate the contribution of sCD13 and its receptor in angiogenesis- and leukocyte-dependent diseases. sCD13 and B1R may be potential therapeutic targets in RA and other diseases.
(Public Health Relevance) Rheumatoid arthritis (RA) is a chronic inflammatory disease in which monocyte (MN) recruitment into the sites of inflammation and new blood vessel formation (angiogenesis) play an important role in the pathogenesis of RA. In this study, we will examine the contribution of aminopeptidase N (CD13) and its receptor, B1R, in angiogenesis and MN ingress in RA and models of RA.