Studies on target molecules for anti-endothelial cells autoantibodies detected in systemic vasculitis.

为了了解系统性血管炎的病理生理学，识别和表征抗内皮细胞抗体（AECA）的自身抗原（autoAgs）至关重要，AECA在血管炎中经常被检测到。为此，我们使用了蛋白质组学方法。具体地说，我们分别从人脐静脉内皮细胞（HUVEC）和Hela细胞中提取蛋白质作为参考，通过二维电泳（2DE）进行分离。然后，我们用Western印迹法（WB）检测了系统性血管炎患者血清中识别的HUVEC特异性蛋白。此外，我们通过肽质量指纹图谱和MS/MS分析鉴定了AECA的候选autoAgs。制备所鉴定的autoAgs的重组蛋白，我们研究了它们的临床意义。结果，我们检测到50个AECA的候选autoAg。其中一种鉴定的autoAg是过氧化物氧还蛋白II（Prx II）。在血管炎患者中，抗Prx Ⅱ自身抗体阳性率为68%，在无明显血管炎的胶原病患者中，抗Prx Ⅱ自身抗体阳性率为7.3%，在健康对照组中，抗Prx Ⅱ自身抗体阳性率为4.0%。临床上，抗Prx II自身抗体的存在与凝血酶-抗凝血酶复合物（达特）和D-二聚体水平升高相关。此外，在HUVEC表面检测到Prx II。此外，自身抗体的Prx II经常检测到大尺寸血管炎，如乳腺炎综合征。总之，我们通过蛋白质组学方法证明了Prx II是AECA的靶点之一。抗Prx II抗体主要在血管炎性疾病中检出，将成为血管炎的新标志物。

To understand pathophysiology of systemic vasculitis, it would be critical to identify and characterize autoantigens (autoAgs) for the anti-endothelial cell antibody(AECA), which are detected frequently in vasculitis. For this aim, we used proteomics approaches. Specifically, we separated proteins extracted from human umbilical vein endothelial cells(HUVEC) and Hela cells as a reference respectively by 2-dimentional electrophoresis(2DE). We then detected HUVEC-specific proteins which were recognized by serum samples from patients with systemic vasculitis by western blotting(WB). Further, we identified the candidate autoAgs for AECA by peptide mass fingerprinting and the MS/MS analysis. Preparing recombinant proteins for the identified autoAgs, we investigated their clinical significance. As a result, we detected 50 candidate autoAgs for AECA. One of the identified autoAgs was peroxiredoxin II(Prx II). The autoantibodies (autoAbs) to Prx II were detected in 68% of the tested patients with vasculitis, 7.3% in tested patients with collagen diseases but no appearent vasculitis, and 4.0% in healthy controls. Clinically, presence of the autoAbs to Prx II was linked to the elevated levels of the thrombin-antithrombin complex(TAT) and D-dimer. In addition, Prx II was detected on the surface of HUVEC. Further, the autoAbs to Prx II were frequently detected in large-size vasculitis like aortitis syndrome. Taken, together, we demonstrated by the proteomic approach that Prx II is one of the targets of AECA. The autoAb to Prx II, detected predominantly in diseases with vasculitis, would be a new marker for presence of vasculitis.