Preeclampsia is a hypertensive disorder of pregnancy that is a leading cause of maternal and fetal morbidity and mortality. The cause of preeclampsia is not known, but the pregnancy specific expression of protease- activated receptor-1 (PAR-1) on neutrophils may hold important keys to understanding the origins of the disease and the underlying causes of maternal organ dysfunction. We propose a novel pathway through which elevated levels of circulating proteases in preeclamptic women activate neutrophil PAR-1, which in turn activates RhoA kinase (ROCK), which triggers translocation of TET2 (tet methylcytosine dioxygenase) and inflammatory transcription factors, such as NF-?B, into the nucleus resulting in specific changes in DNA methylation that cause alterations in expression of genes involved in inflammation. This would be a pregnancy specific inflammatory mechanism because PAR-1 is only expressed on neutrophils during pregnancy. Given the extensive vascular infiltration of neutrophils in preeclampsia, this could explain vascular dysfunction leading to clinical manifestation of symptoms.
In Aim 1 we will determine if proteases activate pregnancy neutrophils by a PAR-1, ROCK pathway to epigenetically regulate inflammatory genes via enzymatic de-methylation of DNA by TET2, the TET protein expressed in leukocytes. We will determine if TET2 nuclear translocation coincides with NF-?B, and if the inflammatory response induced by proteases or present in neutrophils of preeclamptic women can be prevented by inhibition of PAR-1 or ROCK. By using a TET2 knockout cell line, we will determine if the expression of inflammatory genes requires TET2 activation.
In Aim 2 we will determine epigenetic alterations present in neutrophils of preeclamptic women that are due to TET2. We will identify inflammatory gene loci de-methylated by TET, and then determine if de-methylation of these loci opens up inflammatory transcription factor binding sites as a mechanism for increased gene expression.
In Aim 3 we will evaluate if proteases activate TET2 in neutrophils of normal pregnant women resulting in a pattern for inflammatory genes mimicking preeclampsia. These experiments will provide evidence that elevated levels of circulating proteases in preeclampsia could mediate the in vivo activation of neutrophils. These studies will provide new information on pregnancy specific activation of neutrophils to mediate inflammatory response, and may provide new strategies by identifying a molecular target for the treatment of preeclampsia with PAR-1 inhibitors.
Preeclampsia is a hypertensive disorder of pregnancy that occurs in 5-7% of pregnancies and is a leading cause of maternal and fetal morbidity and mortality. This application will evaluate if the pregnancy specific expression of protease-activated receptor-1 (PAR-1) on neutrophils mediates inflammation by being activated by elevated levels of proteases present in the circulation of preeclamptic women. If so, these studies may provide new strategies for treatment of preeclampsia with PAR-1 inhibitors by identifying a key molecular target.
