Background/Purpose �:The mechanisms of the blood vessel injury in giant cell arteritis (GCA), a systemic vasculitis characterized by inflammation of large- and medium-sized vessels, remain to be fully solved. Anti-endothelial cell antibodies (AECA) are antibodies that are detected frequently in vasculitis, including GCA. However, AECA target molecules have been poorly incompletely identified, which hampers our understanding the roles of AECA in disease pathogenesis. Methods �:We identified target antigens for AECA in patients with GCA using agarose 2-dimensional electrophoresis (agarose 2DE) and WB followed by mass spectrometry and accessed to pathophysiological roles in GCA. To detect antigens recognized by GCA sera predominantly in extracted proteins from endothelial cells (EC-specific proteins), the results of agarose 2DE-WB using human aortic endothelial cells (HAEC) were compared with that using human aortic adventitial fibroblasts. Furthermore, to detect antigens on HAEC recognized predominantly in sera with patients with GCA (GCA-specific proteins), the results of 2DE-WB using sera from GCA were compared with that using sera from healthy donors. Results: A total of 31 proteins identified from 23 protein spots recovered from 2DE gel were determined successfully. Three proteins, Ezrin, Zyxin and FK506-binding protein 4, were EC-specific proteins as well as GCA-specific proteins. Gene Ontology analysis showed the EC-specific protein group was mainly classified into the metabolism and the defense and immunity group. In the GCA-specific protein group, the proteins were classified into broad functional categories except for metabolism group. By IPA analysis, more than half of the identified proteins were closely related ubiquitin. Regarding relationships between the identified proteins and cytokines, chemokines, and adhesion molecules, TNF-α was also involved in this signaling network. Using both proteomics and immunocytochemical analyses, we report here for the first time the identification of a specific target antigen for AECA in GCA, zyxin, a focal adhesion protein. Anti-zyxin antibodies were detected in 37% of patients with GCA and in 15% of patients with Takayasu’s arteritis but not in healthy controls. Interestingly, the titer of anti-zyxin antibodies tended to be higher in all untreated patients with GCA than in treated patients. Half of GCA patients with polymyalgia rheumatica had antibodies to zyxin. Using immunocytochemistry analysis, we observed that zyxin translocated from cytoplasm or membrane to the nucleus in ECs stimulated with TNF-α and IL-1β, respectively. Using zyxin siRNA knockdown, zyxin mainly regulates IL-8 production from ECs stimulated with TNF-α and IL-1β, respectively. Furthermore, the increased IL-8 production via zyxin from ECs was inhibited by treatment with glucocorticoids and treatment with anti-zyxin antibodies. Conclusion �:Zyxin is a target antigen for AECA in GCA. Because zyxin is involved in regulating inflammatory responses in ECs via its translocation to the nucleus, the presence of anti-zyxin antibodies in GCA strongly implicates these antibodies in the pathogenesis of GCA or in disease progression.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/novel-roles-for-zyxin-in-the-pathogenesis-of-giant-cell-arteritis/