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，是母婴发病率的主要原因。 和死亡率。先兆子痫的原因尚不清楚，但妊娠特异表达的蛋白酶- 中性粒细胞上活化的受体-1(PAR-1)可能是理解 疾病和产妇器官功能障碍的根本原因。我们提出了一种新的途径，通过它 先兆子痫患者循环中的蛋白水解酶水平升高会激活中性粒细胞PAR-1，进而 激活RhoA激酶(ROCK)，从而触发TET2(tet甲基胞嘧啶双加氧酶)的易位和 炎性转录因子，如核因子-DNAB，进入细胞核，导致的特殊变化 引起炎症相关基因表达变化的甲基化。这将是一次怀孕 特殊的炎症机制，因为PAR-1只在怀孕期间的中性粒细胞上表达。vt.给出 先兆子痫患者中性粒细胞广泛的血管浸润，这可以解释导致血管功能障碍的原因。 以临床症状表现为主。在目标1中，我们将确定蛋白酶是否通过以下方式激活妊娠中性粒细胞 通过DNA酶促去甲基化对炎症基因进行表观调控的PAR-1，ROCK途径 通过TET2，Tet蛋白在白细胞中表达。我们将确定TET2核转位是否重合 对于NF-B，以及由蛋白酶诱导的或存在于先兆子痫患者中性粒细胞中的炎症反应 女性可以通过抑制PAR-1或ROCK来预防。通过使用TET2基因敲除细胞系，我们将 确定炎症基因的表达是否需要TET2的激活。在目标2中，我们将确定 由TET2引起的子痫前期妇女中性粒细胞存在表观遗传学改变。我们将确定 用Tet去甲基化炎症基因座位，然后确定这些座位的去甲基化是否开启 炎性转录因子结合部位作为基因表达增加的机制。在《目标3》中，我们将 评估蛋白水解酶是否激活正常孕妇中性粒细胞中的TET2，从而导致 模仿先兆子痫的炎症基因。这些实验将提供证据，证明高水平的 子痫前期患者循环中的蛋白水解酶可介导中性粒细胞的体内活化。这些研究将 提供妊娠特异性中性粒细胞激活以介导炎症反应的新信息 可能通过确定PAR-1治疗子痫前期的分子靶点来提供新的策略 抑制剂。

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.