SUMMARYWhile it has been claimed that some anti-endothelial cell antibodies (AECA) activate EC, there is also evidence that others trigger apoptosis. To address the issue of whether activation is a prerequisite for AECA-mediated apoptosis of EC, 23 AECA-positive sera were evaluated for their ability to induce activation and/or apoptosis. Activation was defined as an over-expression of E-selectin and intercellular adhesion molecule 1. Optical microscopy, annexin V binding, hypoploid cell enumeration, and determination of poly (ADP-ribose) polymerase cleavage-related products were used to assess apoptosis. Four functional profiles were defined: 10 sera promoted activation and apoptosis (act1/apo1), one was act1/apo-, six act-/apo1, and the remaining six act-/apo-. The reduced membrane expression of thrombomodulin was associated with apoptosis, rather than activation. Caspase-3 was implicated in the two models of apoptosis, the ratios of several survival proteins to Bax decreased, regardless of the ability of apo1 AECA to activate the cells, while radical oxygen species did not appear to be involved. Furthermore, it occurred that macrophages engulfed EC treated with apoptosis-promoting AECA, but not those incubated with AECA that did not induce apoptosis. Hence, AECA represent an extremely heterogeneous family of autoantibodies, not only because of the variety of their target antigens, but also the subsequent diversity of their effects.

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Since false-negative AECA may result from lack of expression of various antigens on a specific EC, analysis of AECA in SSc patients requires using several EC types, including microvascular EC.

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As endothelial cells (EC) express heparin-like glycosaminoglycans, such as heparan sulfate, it was essential to investigate the relation of anti-EC antibody (AECA) to heparin reactivity. AECA were detected in 43 of 131 autoimmune sera and anti-heparin antibodies (AHA) in 25. These autoimmune reactivities were significantly associated (P corrected < 0.0005). Seven AECA-positive/AHA-positive and three AECA-negative/AHA-positive sera were affinity-purified using protein G column followed by a heparin-Sepharose column. Two populations of AECA were recovered from the second column. One was eluted with 0.4 M NaCl which bound to EC and to solid-phase heparin with low affinity, but not to soluble heparin. The second population of AECA, which was eluted with 4 M guanidine HCl/2 M NaCl, recognized EC and solid-phase heparin with high affinity, but also soluble heparin. The latter population of AECA might thus be an important cause of autoimmune vascular thrombosis in systemic diseases.