AT1 receptor agonistic antibodies from preeclamptic patients cause vascular cells to express tissue factor

Abstract

Background - We recently described autoantibodies (angiotensin-1 receptor autoantibodies, AT1-AA) directed at the AT1 receptor in the serum of preeclamptic patients, whose placentas are commonly infarcted and express tissue factor (TF). Mechanisms of how AT1-AA might contribute to preeclampsia are unknown. We tested the hypothesis that AT1-AA cause vascular smooth muscle cells (VSMC) to express TF. Methods and Results - IgG from preeclamptic patients containing AT1-AA was purified with anti-human IgG columns. AT1-AA were separated from the IgG by ammonium sulfate precipitation. We transfected Chinese hamster ovary cells overexpressing the AT1 receptor with TF promoter constructs coupled to a luciferase reporter gene. VSMC were obtained from human coronary arteries. Extracellular signal- related kinase activation was detected by an in-gel kinase assay. AP-1 activation was determined by electromobility shift assay. TF was measured by ELISA and detected by immunohistochemistry. Placentas from preeclamptic women stained strongly for TF, whereas control placentas showed far less staining. We proved AT1-AA specificity by coimmunoprecipitating the AT1 receptor with AT1-AA but not with nonspecific IgG. Angiotensin (Ang) II and AT1-AA both activated extracellular signal-related kinase, AP-1, and the TF promoter transfected VSMC and Chinese hamster ovary cells, but only when the AP-1 binding site was present. We then demonstrated TF expression in VSMC exposed to either Ang II or AT1-AA. All these effects were blocked by losartan. Nonspecific IgG or IgG from nonpreeclamptic pregnant women had a negligible effect. Conclusions - We conclude that AT1-AA and Ang II both stimulate the AT1 receptor and initiate a signaling cascade resulting in TF expression. These results show an action of AT1-AA on human cells that could contribute to the pathogenesis of preeclampsia.