Background/Purpose: Juvenile dermatomyositis (JDM), the most common inflammatory myopathy of childhood, is a rare systemic autoimmune vasculopathy. Juvenile idiopathic arthritis (JIA), the most common pediatric rheumatic disease, is a heterogeneous group of inflammatory diseases. The etiology of these diseases is unknown, and we lack disease and disease activity–specific biomarkers. Anti-endothelial cell antibodies (AECA) are autoantibodies against antigens on endothelial cell surface and are detected frequently in rheumatic diseases such as vasculitis. We tried to detect target proteins of AECA comprehensively by proteomics in patients with pediatric rheumatic diseases such as JIA and JDM. Furthermore we investigated relationship to these diseases in AECA from individual identified target antigens.

Methods: We separated proteins extracted from human aortic endothelial cells (HAEC) by one-dimensional electrophoresis (1DE) and two-dimensional electrophoresis (2DE) and transferred them onto membranes. By western blotting (WB) using sera from patients with pediatric rheumatic diseases and healthy donors, we detected antigens that were positive only in pediatric rheumatic disease sera but not in healthy donor sera. We next identified the detected proteins by peptide mass finger-printing.

Results: By using 1DE, 70-kDa and 88-kDa target protein of AECA were detected only in active patients without treatment. 48-kDa and 75-kDa target protein of AECA were detected only in active patients with treatment. 110-kDa target protein of AECA was detected only in active patients. These target proteins of AECA were not detected in inactive patients and healthy donors. Eighteen candidate protein spots as pediatric rheumatic diseases-specific proteins were detected in 2DE-WB.  From these spots, we successfully identified 738 proteins which we functionally characterized�: (1) 37% were ATP-related proteins such as proteins of the tricarboxylic acid (TCA) cycle (e.g., phosphoserine aminotransferase and methylmalonyl-CoA mutase; (2)19% were muscle-related proteins, including tropomyosin beta chain and PDZ and LIM domain protein 7; (3)13% were calcium regulated protein and/or calcium binding proteins (e.g., annexin A1 and calreticulin);  (4)10% were redox related proteins (e.g., procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2, and procollagen-lysine,2-oxoglutarate 5-dioxygenase 2). Furthermore 50% of the identified antigens represented membrane proteins.

Conclusion: Antibodies directed toward the proteome of HAEC are present in the sera of patients with pediatric rheumatic diseases. These antibodies recognize a broad range of cellular targets, including those associated with skeletal muscle function and inflammatory processes.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-target-antigens-for-anti-endothelial-cell-antibodies-in-patients-with-pediatric-rheumatic-diseases-using-proteomics/