High Throughput Strategies for Preeclampsia Therapy

先兆子痫治疗的高通量策略

基本信息

批准号：
9035207
负责人：
Derek S Boeldt
金额：
$ 7.57万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-01 至 2018-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9035207
关键词：
Accounting Adult Affect Arteries Biological Assay Birth Blood Pressure Blood Vessels Cardiovascular Diseases Cell Line Cells Clinical Clinical Trials Conjugated Linoleic Acids Connexin 43 Data Diabetes Mellitus Dietary Intervention Disease Edema Electrical Resistance Emergency Situation Endocrine Endothelial Cells Endothelium Failure Family Fetal Growth Retardation Fetus Food Functional disorder Future Goals Growth Health Hormones Human Hypertension In Vitro Incidence Intercellular Junctions Isomerism Libraries Life Ligands Maternal Mortality Measures Mediating Methods Modeling Molecular Monitor Mothers Nifedipine Nutraceutical Obesity Pathway interactions Pharmaceutical Preparations Phosphorylation Phosphotransferases Population Pre-Eclampsia Pregnancy Production Protein Isoforms Proteins Proteinuria Reader Recovery Regulation Resistance Risk Signal Transduction Symptoms System Techniques Therapeutic Thinking Time Umbilical Cord Blood United States Vasodilation Vasodilator Agents Work base cytokine electric impedance endothelial dysfunction fetal high throughput screening imaging modality inhibitor/antagonist innovation maternal hypertension minimal risk monolayer novel novel therapeutics offspring public health relevance receptor research study response screening sound src-Family Kinases temporal measurement translational study unborn child

项目摘要

DESCRIPTION (provided by applicant): Preeclampsia is a gestational disorder characterized by maternal hypertension, often with proteinuria, affecting up to 10% of all pregnancies worldwide leading to maternal mortality in extreme cases. In the United States, PE affects roughly 3-5% of pregnancies and has increased 25% over the last 20 years. Fetal complications often include intrauterine growth restriction and increased incidence of cardiovascular disorders throughout life. At the endothelial level, the loss of vasodilator production and further ongoing decline in monolayer integrity largely account for the observed symptoms of hypertension and edema/proteinuria, respectively. Work from our own lab suggests that loss of Cx43 function by direct phosphorylation underlies the immediate loss of vasodilation response while others have shown longer term progressive breakdown of cell-cell junctions may be responsible for further edema/proteinurea. Of note, both/all these responses are known to be mediated by Src family kinases sensitive to the drug PP2. Treatment of preeclampsia is difficult in part because of a limited understanding of the origin of the disease and the failure to identify a single consistentl responsible endocrine factor. We have recently pointed out a critical convergence point of the many potential negative growth factors and cytokines at a post receptor level is co-regulation of Src. Limited preliminary data showing PP2 can rescue endothelial cell dysfunction in vitro in response to multiple ligands supports the hypothesis. We now propose to develop high throughput methods to 1) verify our hypothesis that the molecular co-regulation of endothelial dysfunction at the level of Cx43 and cell-cell junctions by growth factors and cytokines is through a common Src (PP2 sensitive) pathway, and 2) screen a novel family of nutraceutical compounds which are safe for human use and may have therapeutic relevance in preeclampsia. Aim 2 in particular follows from preliminary experiments showing the natural food product conjugated linoleic acid (CLA) in the 10,12 isoform, a Src inhibitor, can rescue inhibited Ca2+ signaling function in our ovine model. We further hypothesize that 10,12 and other CLA isoforms will recover both Ca2+ responses and monolayer integrity in HUVEC otherwise inhibited by growth factors and cytokines. A major limitation to progress is the Ca2+ imaging method used to date is time consuming, so screening of multiple hormones and drugs is impractical. The electrical cell substrate impedance sensing (ECIS) system measures electrical resistance across a cell monolayer in real time, and provides a high resolution temporal view of cell-cell junction breakdown in multiwell format. Thus, the proposed aims will provide a two- pronged, high throughput screening approach for both initiation and recovery of endothelial dysfunction at the levels of vasodilatory function and monolayer integrity. To that end: Specific Aim 1 screens growth factors and cytokines for the ability to inhibit Cx43 mediated Ca2+ signaling in HUVEC and monolayer junctional integrity. Specific Aim 2 screens CLA isomers for their ability to rescue Ca2+ signaling and monolayer integrity function in HUVEC.

描述（由申请人提供）：先兆子痫是一种以孕产妇高血压为特征的妊娠疾病，通常伴有蛋白尿，影响全世界多达 10% 的妊娠，在极端情况下导致孕产妇死亡，在美国，PE 影响大约 3-5%。妊娠率在过去 20 年中增加了 25%，胎儿并发症通常包括宫内生长受限和一生中心血管疾病的发生率增加。血管舒张剂的产生和单层完整性的进一步持续下降分别在很大程度上解释了观察到的高血压和水肿/蛋白尿症状，我们自己实验室的工作表明，直接磷酸化引起的 Cx43 功能丧失是血管舒张反应立即丧失的基础，而其他反应则表现出更长的时间。细胞与细胞连接的进行性破坏可能是导致进一步水肿/蛋白尿的原因。值得注意的是，已知这两种/所有这些反应都是由对药物敏感的 Src 家族激酶介导的。 PP2.先兆子痫的治疗很困难，部分原因是对疾病起源的了解有限，并且未能确定单一一致的内分泌因子。我们最近指出了许多潜在负生长因子和细胞因子的关键汇聚点。有限的初步数据显示 PP2 可以响应多种配体而在体外挽救内皮细胞功能障碍，这支持了这一假设。假设生长因子和细胞因子在 Cx43 和细胞-细胞连接水平上对内皮功能障碍的分子共同调节是通过共同的 Src（PP2 敏感）途径进行的，并且 2）筛选了一个新的营养化合物家族，这些化合物对于目标 2 特别是根据初步实验得出的，该实验显示天然食品产品共轭亚油酸 (CLA) 的 10,12 同种型（一种）。 Src 抑制剂可以挽救我们的绵羊模型中受抑制的 Ca2+ 信号传导功能，我们进一步指出，10,12 和其他 CLA 亚型将恢复 HUVEC 中的 Ca2+ 反应和单层完整性，否则会受到生长因子和细胞因子的抑制。迄今为止使用的 Ca2+ 成像方法非常耗时，因此筛选多种激素和药物是不切实际的。细胞基质阻抗传感 (ECIS) 系统实时测量细胞单层的电阻。因此，所提出的目标将为血管舒张功能和恢复水平的内皮功能障碍提供双管齐下的高通量筛选方法。为此：Specific Aim 1 筛选生长因子和细胞因子抑制 HUVEC 中 Cx43 介导的 Ca2+ 信号传导的能力，而单层 Specific Aim 2 筛选 CLA 异构体的能力。他们能够挽救 HUVEC 中的 Ca2+ 信号传导和单层完整性功能。