Proteases Activate Pregnancy Neutrophils by a Protease-Activated Receptor 1 Pathway: Epigenetic Implications for Preeclampsia.

We tested a novel hypothesis that elevated levels of proteases in the maternal circulation of preeclamptic women activate neutrophils due to their pregnancy-specific expression of protease-activated receptor 1 (PAR-1). Plasma was collected longitudinally from normal pregnant and preeclamptic women and analyzed for MMP-1 and neutrophil elastase. Neutrophils were isolated for culture and confocal microscopy. Omental fat was collected for immunohistochemistry. Circulating proteases were significantly elevated in preeclampsia. Confocal microscopy revealed that tet methylcytosine dioxygenase 2 (TET2), a DNA de-methylase, and p65 subunit of NF-κB were strongly localized to the nucleus of untreated neutrophils of preeclamptic women, but in untreated neutrophils of normal pregnant women they were restricted to the cytosol. Treatment of normal pregnancy neutrophils with proteases activated PAR-1, leading to activation of RhoA kinase (ROCK), which triggered translocation of TET2 and p65 from the cytosol into the nucleus, mimicking the nuclear localization in neutrophils of preeclamptic women. IL-8, an NF-κB-regulated gene, increased in association with TET2 and p65 nuclear localization. Co-treatment with inhibitors of PAR-1 or ROCK prevented nuclear translocation and IL-8 did not increase. Treatment of preeclamptic pregnancy neutrophils with inhibitors emptied the nucleus of TET2 and p65, mimicking the cytosolic localization of normal pregnancy neutrophils. Expression of PAR-1 and TET2 were markedly increased in omental fat vessels and neutrophils of preeclamptic women. We conclude that elevated levels of circulating proteases in preeclamptic women activate neutrophils due to their pregnancy-specific expression of PAR-1 and speculate that TET2 DNA de-methylation plays a role in the inflammatory response.

Matrix Metalloprotease-1 and Elastase Are Novel Uterotonic Agents Acting Through Protease-Activated Receptor 1.

Matrix metalloproteinase-1 (MMP-1) and neutrophil elastase are proteolytic enzymes involved in tissue remodeling, but a role for them as uterotonic agents has not been considered. However, both these proteases activate protease-activated receptor 1 (PAR-1) that mediates thrombin-induced contractions. Matrix metalloproteinase-1 and elastase are products of neutrophils that infiltrate intrauterine tissues at the time of labor, so we tested the hypothesis that these proteases might be novel uterotonic agents acting via PAR-1. Decidual tissue was collected from fetal membranes of term not-in-labor (TNL), term labor (TL), and preterm labor (PTL) women and analyzed for gene and protein expression of MMP-1 and neutrophil elastase. Contractile effects of MMP-1 and elastase were tested with uterine strips of day 19 and 20 gestation rats. Expression of MMP-1 and neutrophil elastase was increased in TL and PTL as compared to TNL. Expression of both the pro- and active enzymes of MMP-1 increased progressively from TNL to TL to PTL. Tumor necrosis factor-α, a neutrophil product, increased MMP-1 in decidual and myometrial cells. Both MMP-1 and elastase stimulated strong contractions of myometrial strips, which were prevented by inhibition of PAR-1 and inhibition of inositol trisphosphate receptor or calcium channel blockade. Indomethacin did not prevent protease-induced contractions. These data suggest that MMP-1 and neutrophil elastase may be important but heretofore unrecognized players in stimulating uterine contractions at the time of labor, and they may explain why indomethacin delays, but does not prevent, PTL because indomethacin inhibits the prostaglandin component but not the protease component of labor.

Mechanisms of enhanced vascular reactivity in preeclampsia.

Preeclamptic women have enhanced blood pressure response to angiotensin II and extensive systemic vascular infiltration of neutrophils. Neutrophils release reactive oxygen species that might activate the RhoA kinase pathway to enhance vascular reactivity. We hypothesized that enhanced vascular reactivity in preeclampsia is attributed to neutrophil-mediated reactive oxygen species activation of the RhoA kinase pathway. Omental arteries were obtained at cesarean section and studied using a myograph system. We found that arteries of preeclamptic women had extensive infiltration of neutrophils and enhanced reactivity to angiotensin II. Treatment of arteries of normal pregnant women with reactive oxygen species or activated neutrophils enhanced vessel reactivity to angiotensin II mimicking preeclamptic vessels. Pretreatment with superoxide dismutase/catalase to quench reactive oxygen species or RhoA kinase inhibitor blocked enhanced responses in preeclamptic and normal vessels. Reactive oxygen species also enhanced vessel reactivity to norepinephrine, which was blocked by RhoA kinase inhibition. Treatment of arteries with reactive oxygen species increased RhoA kinase activity 3-fold, whereas culture of human vascular smooth muscle cells with angiotensin II and activated neutrophils or reactive oxygen species resulted in phosphorylation of key proteins in the RhoA kinase pathway. We conclude that enhanced vascular reactivity of omental arteries in preeclampsia is attributed to reactive oxygen species activation of the RhoA kinase pathway and that enhanced vascular reactivity is likely attributed to the infiltration of neutrophils. We speculate that neutrophil infiltration into systemic vasculature of preeclamptic women is an important mechanism for hypertension.