Aspirin Inhibits the Inflammatory Response of Protease-Activated Receptor 1 in Pregnancy Neutrophils: Implications for Treating Women with Preeclampsia

Abstract

Neutrophils expressing cyclooxygenase-2 (COX-2) extensively infiltrate maternal blood vessels in preeclampsia, associated with vascular inflammation. Because pregnancy neutrophils also express protease-activated receptor 1 (PAR-1, F2R thrombin receptor), which they do not in non-pregnant subjects, they can be activated by proteases. We tested the hypothesis that aspirin at a dose sufficient to inhibit COX-2 would reduce inflammatory responses in preeclampsia neutrophils. Neutrophils were isolated from normal pregnant and preeclamptic women at approximately 30 weeks’ gestation. Normal pregnancy neutrophils were treated with elastase, a protease elevated in preeclampsia, or elastase plus aspirin to inhibit COX-2, or elastase plus pinane thromboxane, a biologically active structural analog of thromboxane and a thromboxane synthase inhibitor. Preeclamptic pregnancy neutrophils were treated with the same doses of aspirin or pinane thromboxane. Confocal microscopy with immunofluorescence staining was used to determine the cellular localization of the p65 subunit of nuclear factor-kappa B (NF-κB) and media concentrations of thromboxane were measured to evaluate the inflammatory response. In untreated neutrophils of normal pregnant women, p65 was localized to the cytosol. Upon stimulation with elastase, p65 translocated from the cytosol to the nucleus coincident with increased thromboxane production. When neutrophils were co-treated with aspirin or pinane thromboxane, elastase was not able to cause nuclear translocation of p65 or increase thromboxane. In untreated neutrophils of preeclamptic women, the p65 subunit was present in the nucleus and thromboxane production was elevated, but when preeclamptic neutrophils were treated with aspirin or pinane thromboxane, p65 was cleared from the nucleus and returned to the cytosol along with decreased thromboxane production. These findings suggest that COX-2 is a downstream mediator of PAR-1 and demonstrate that PAR-1- mediated inflammation can be inhibited by aspirin. Given the extensive and ubiquitous expression of PAR-1 and COX-2 in preeclamptic women, consideration should be given to treating women with preeclampsia using a dose of aspirin sufficient to inhibit COX-2.