Presentation: Identification of Targets of the Anti-Endothelial Cell Antibodies in Patients with Systemic Vasculitis: By a Proteomic Approach (2007)

2011 Identification of Targets of the Anti-Endothelial Cell Antibodies in Patients with Systemic Vasculitis: By a Proteomic Approach

PURPOSE Anti-endothelial cell antibodies (AECA) are thought to be involved in pathophysiology of systemic vasculitis. However, target molecules of AECA have been poorly identified, which hampers understanding of roles of AECA in detail. We here tried to detect and identify target proteins of AECA comprehensively by proteomics. Further, we investigated clinical importance of the identified autoantigens and their related proteins by ELISA and western blotting using by a large number of serum samples from patients with or without vasculitis.
METHODS To detect endothelial cell-specific autoantigens for AECA, we separated proteins extracted from human umbilical cord vein endothelial cell (HUVEC) and HeLa cell respectively by 2-dimensional electrophoresis(2DE) and then transferred them onto membranes, which were subjected to western blotting using serum samples from patients with systemic vasculitis. We selected autoantigenic spots that were detected only in the HUVEC sample but not in the HeLa cell sample, which should be candidates for HUVEC-specific autoantigens. We next identified the detected proteins by peptide mass finger-printing (PMF) and characterized antigenecity by preparing recombinant autoantigens and antibodies to them. Finally, we compared the clinical parameters between the autoantibodies-positive and negative patients to assess clinical importance of the antibodies.
RESULTS We detected about 150 HUVEC-specific autoantigens recognized by serum samples from patients with vasculitis. We have identified 63 proteins out of the detected proteins by PMF so far. One of the identified proteins was found peroxiredoxin2 (Prx2), an anti-oxidative enzyme. The autoantibodies to Prx2 were detected in 61% of the patients with vasculitis, in particular, 88% in patients with Takayasu’s diease, but in 4.0% of healthy controls tested and were found significantly higher in the patients with large vessel vasculitis than middle and small vessel one. Further, indirect immunofluorecence stainig revealed existence of Prx2 on the cell surface of HUVEC and WB using cell lysate proved expression of Prx2 not only in HUVEC but also in HAEC, HPAEC, HMVEC and HMVEC-L. The anti-Prx2 antibodies also increased inflammatory cytokine secretion significantly, which included IL-6, IL-1β, GROα, IL-8, GM-CSF, IL-1ra and MCP-1. Clinically, titers of D-dimer and TAT were found significantly higher in the anti-Prx2 positive patients with vasculitis than the negative ones. Furthermore anti-Prx2 titers tended to change in parallel with activity of the vasculitis.
CONCLUSIONS The autoantibodies to Prx2, which we found for the first time, would be a useful diagnostic marker for systemic vasculitis. Pathologically, binding of the anti-Prx2 antibodies to Prx2 on the surface of endothelial cells may cause vascular dysfunction.

 R. Karasawa, None; S. Ooka, None; K. Masuko, None; S. Ozaki, None; K. Nishioka, None; K. Yudoh, None; T. Kato, None.