In vivo analysis of the apoptosis‐inducing effect of anti–endothelial cell antibodies in systemic sclerosis by the chorionallantoic membrane assay

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Abstract
Objective Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology characterized by mononuclear cell infiltration and fibrosis. Vascular injury occurs early in the course of disease, and previous in vitro studies suggest a primary role for anti–endothelial cell antibodies (AECAs) in mediating endothelial cell apoptosis. The aim of the present study was to analyze the apoptosis‐inducing effect of AECAs in vivo. Methods The optimum animal model for transfer experiments was the University of California at Davis line 200 (UCD‐200) chickens that spontaneously develop a hereditary disease with features closely resembling those of scleroderma in humans. AECA‐positive serum samples from UCD‐200 chickens were used for intravenous injection into normal CC chicken embryos on embryonic day (ED) 13 as well as for application onto chorionallantoic membranes (CAMs) of healthy control lines on ED 10. CAMs of ED 16 embryos and combs of 1‐week‐old CC chickens that had received the injected serum samples were analyzed for apoptotic endothelial cells by TUNEL. Results Staining of frozen CAM sections by immunofluorescence showed evidence of in vivo binding of AECAs to the microvascular endothelium. In most groups, transfer of AECA‐positive sera resulted in a significant increase in endothelial cell apoptosis as compared with controls. Conclusion This study is the first to demonstrate the in vivo apoptosis‐inducing effects of AECAs. The findings support our hypothesis of a primary pathogenetic role of AECAs in SSc.