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

先兆子痫中 PAR-1 和 TET2 的妊娠特异性蛋白酶激活——对治疗的影响

基本信息

批准号：
10190981
负责人：
SCOTT W WALSH
金额：
$ 31.57万
依托单位：
VIRGINIA COMMONWEALTH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10190981
关键词：
Binding Sites Blood Circulation Blood Vessels Cell Line Cell Nucleus Clinical DNA Methylation Dioxygenases Disease Epigenetic Process Functional disorder Gene Expression Gene Expression Profile Gene Targeting Genes Inflammation Inflammatory Inflammatory Response Knock-out Leukocytes Mediating Methylation Molecular Target Morbidity - disease rate NF-kappa B Neutrophil Activation Neutrophil Infiltration Nuclear Nuclear Translocation Organ PAR-1 Receptor Pathway interactions Pattern Peptide Hydrolases Phosphotransferases Pre-Eclampsia Pregnancy Pregnant Women Prevention approach Proteins Signs and Symptoms Symptoms Testing Tetanus Helper Peptide Vascular Diseases Woman experimental study fetal genomic locus in vivo inhibitor/antagonist molecular targeted therapies mortality neutrophil novel novel strategies pregnancy disorder prevent transcription factor treatment strategy

项目摘要

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.

先兆子痫是一种妊娠期高血压疾病，是母婴发病的主要原因和死亡率。先兆子痫的原因尚不清楚，但蛋白酶的妊娠特异性表达中性粒细胞上的激活受体 1 (PAR-1) 可能是了解中性粒细胞起源的重要关键。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女性循环蛋白酶水平升高会激活中性粒细胞 PAR-1，进而激活中性粒细胞 PAR-1 激活 RhoA 激酶 (ROCK)，触发 TET2（tet 甲基胞嘧啶双加氧酶）易位，炎症转录因子，如 NF-κB，进入细胞核，导致 DNA 发生特定变化甲基化会导致炎症相关基因表达的改变。这将是怀孕特定的炎症机制，因为 PAR-1 仅在妊娠期间在中性粒细胞上表达。给定先兆子痫中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症状的临床表现为主。在目标 1 中，我们将通过以下方式确定蛋白酶是否激活妊娠中性粒细胞： PAR-1、ROCK 通路通过 DNA 酶促去甲基化来表观遗传调节炎症基因 TET2，即白细胞中表达的 TET 蛋白。我们将确定 TET2 核易位是否一致 NF-κB，如果由蛋白酶诱导的炎症反应或存在于先兆子痫的中性粒细胞中女性可以通过抑制 PAR-1 或 ROCK 来预防。通过使用 TET2 敲除细胞系，我们将确定炎症基因的表达是否需要 TET2 激活。在目标 2 中，我们将确定先兆子痫女性的中性粒细胞中存在由 TET2 引起的表观遗传改变。我们将确定通过TET去甲基化炎症基因位点，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，我们将评估蛋白酶是否激活正常孕妇中性粒细胞中的 TET2，从而产生以下模式：模仿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以介导中性粒细胞的体内激活。这些研究将提供关于妊娠特异性激活中性粒细胞介导炎症反应的新信息，以及可能通过确定 PAR-1 治疗先兆子痫的分子靶点来提供新策略抑制剂。