Background/Purpose: Aminopeptidase N (CD13, EC 3.4.11.2) is a metalloproteinase expressed on the surface of fibroblast like synoviocytes (FLS), and as a soluble protein in serum and synovial fluid.  CD13 is also expressed by multiple other cell types including monocytic lineage cells and endothelial cells.  We have shown that CD13 can be released from FLS in culture, and that CD13 is higher in amount and activity in rheumatoid arthritis (RA) synovial fluids compared to osteoarthritis (OA).  The goals of this study are to further explore potential roles for CD13 in the synovium in relation to monocytes and endothelial cells.  

Methods: Synovium samples were cyrosectioned and stained for CD13 (antibody 591.1D7.34) and von Willebrand factor (VWF, rabbit anti-human DAKO) followed by secondary immunoflourescent antibodies.  Co-localization was analyzed with ImageJ.  Chemotaxis was performed in a modified Boyden chamber system using human dermal microvascular endothelial cells (HMVECs) or monocytes isolated from healthy donors.  Chemotaxis was measured toward PBS, recombinant human CD13 (rhCD13) or an enzymatically inactive mutant CD13.  Angiogenesis was measured using a HMVEC tube formation assay and a mouse Matrigel plug assay.

Results: CD13 on endothelial cells has been linked to angiogenesis; however, endothelial CD13 has not been examined in the synovium.   We show that RA synovial sections stained more strongly for CD13 and showed stronger co-localization of CD13 and VWF than either OA or normal samples.  A role for soluble CD13 (sCD13) has also not been determined in endothelial cell or monocyte migration.  We therefore examined whether sCD13 could contribute to angiogenesis and/or monocyte chemotaxis.  We found that CD13 was significantly chemotactic for monocytes over a range from 125ng/ml-500ng/ml CD13 with a peak chemotaxis at 500ng/ml (213.86±43.42 cells/well p=0.05).  sCD13 was strongly chemotactic for HMVECS over a range from 0.5ng/ml-2000ng/ml with peak chemotaxis at 1000ng/ml (164.25±21.13 cells/well, p=0.000173).  The enzymatically inactive mutant of CD13 was also chemotactic for HMVECs (135.02±11.47 cells/well, p=0.012) but to a lesser degree than the enzymatically active form (204.06±24.02 cells/well, p=0.0078).  Depletion of CD13 from RA synovial fluid significantly decreased HMVEC chemotaxis.  Furthermore, rhCD13 significantly increased HMVEC tube formation (p<0.05) in Matrigel, and rhCD13 significantly increased angiogenesis in a mouse Matrigel plug assay (PBS 0.099±0.025 g/dl/mg, CD13 1.49±0.62 g/dl/mg, p<0.05).

Conclusion: Soluble CD13 is shed from FLS and can act as a chemoattractant for T cells and monocytes, bringing these pro-inflammatory cells to the RA joint.  CD13 is present on synovial endothelial cells, but sCD13 can also act as a pro-angiogeneic factor in the synovium in a manner that is partially independent of its enzymatic activity.  The concentrations of CD13 used in these experimental systems correspond to levels of CD13 in the RA joint. In summary CD13 secreted by FLS in the RA joint may contribute to a pro-inflammatory milieu and lead to increased angiogenesis and migration of monocytes and T cells to the RA synovium.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/cd13aminopeptidase-n-contributes-to-angiogenesis-and-monocyte-chemotaxis-in-rheumatoid-arthritis/