Pregnancy Specific Protease Activation of PAR-1 and TET2 in Preeclampsia-Implications for Therapy

先兆子痫是一种妊娠期高血压疾病，是导致孕产妇和胎儿发病的主要原因 and mortality.先兆子痫的原因尚不清楚，但妊娠特异性蛋白酶表达- 中性粒细胞上的活化受体-1（PAR-1）可能是理解这些疾病起源的重要关键。 疾病和母体器官功能障碍的根本原因。我们提出了一种新的途径， 先兆子痫妇女中循环蛋白酶水平升高激活中性粒细胞PAR-1， 激活RhoA激酶（ROCK），其触发TET 2（泰特甲基胞嘧啶双加氧酶）的易位， 炎症转录因子，如NF-κ B B，进入细胞核，导致DNA的特异性变化 甲基化导致炎症相关基因表达的改变。这就是怀孕 由于PAR-1在妊娠期间仅在中性粒细胞上表达，因此PAR-1是特异性炎症机制。给定 在先兆子痫中广泛的中性粒细胞血管浸润，这可以解释血管功能障碍导致 临床症状表现。在目标1中，我们将通过以下方法确定蛋白酶是否激活妊娠中性粒细胞： PAR-1、ROCK通路通过DNA的酶促去甲基化表观遗传调节炎症基因 TET 2是白细胞中表达的泰特蛋白。我们将确定TET 2核易位是否与 与NF-κ B B，如果炎症反应诱导的蛋白酶或目前在中性粒细胞的先兆子痫 女性可以通过抑制PAR-1或ROCK来预防。通过使用TET 2敲除细胞系，我们将 确定炎症基因的表达是否需要TET 2激活。在目标2中，我们将确定 由于TET 2，先兆子痫妇女的中性粒细胞中存在表观遗传改变。我们将确定 炎症基因位点被泰特去甲基化，然后确定这些位点的去甲基化是否开放 炎症转录因子结合位点作为增加基因表达的机制。在目标3中， 评估蛋白酶是否激活正常孕妇中性粒细胞中的TET 2，导致以下模式： 类似先兆子痫的炎症基因这些实验将提供证据表明， 先兆子痫中的循环蛋白酶可介导嗜中性粒细胞的体内活化。这些研究将 提供了关于妊娠特异性激活中性粒细胞介导炎症反应的新信息， 通过鉴定PAR-1治疗先兆子痫的分子靶点， 抑制剂的

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.