Objective
To identify the target molecules of anti-endothelial cell antibodies (AECA), which are thought to be involved in pathophysiology of systemic vasculitis.
Methods
To detect endothelial cell-specific autoantigens for AECA, we separated proteins extracted from human umbilical cord vein endothelial cells (HUVEC) and HeLa cells respectively by 2-dimensional gel electrophoresis (2DE) respectively. They were then transferred onto membranes. By western blotting using serum samples from patients with systemic vasculitis, we detected autoantigens that were positively reacted to the serum samples in the HUVEC samples but not in the Hela cell samples. Then we identified these detected HUVEC-specific autoantigens by mass-fingerprinting. Further, we prepared recombinant autoantigens, by which we confirmed their antigenecity.
Results
We found that 50 protein spots were positively reacted to the serum samples only in the HUVEC proteome, but not in the Hela proteome. These proteins would be candidate autoantigens for AECA. We have identified 6 proteins out of the 50 detected protein by mass fingerprinting so far. One of the proteins was found to be a peroxiredoxin 2, which was an anti-oxidative enzyme. ELISA using a recombinant peroxiredoxin 2revealed that autoantibodies to peroxiredoxin 2 were detected in about 70% of the patients with vasculitis but in very small population of vasculitis-negative autoimmune patients.
Conclusion
Proteomic surveillance is an effective way to identify the targets of AECA. One of the identified autoantibodies, the autoantibody to the peroxiredoxin-2 homologous protein, would have diagnostic and phatophysiolosical importance in the systemic vasculitis.