High Throughput Strategies for Preeclampsia Therapy

先兆子痫治疗的高通量策略

• 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
• 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

 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（由适用提供）：预制是一种妊娠疾病，其特征是母体高血压，通常患有蛋白尿，影响了全球所有怀孕的10％，从而导致极端情况下孕产妇死亡。在美国，PE影响了大约3-5％的怀孕，并且在过去20年中增长了25％。胎儿并发症通常包括内部生长限制和一生中心血管疾病的发生率增加。在内皮水平上，单层完整性的血管扩张产生损失和进一步下降很大程度上分别解释了观察到的高血压和水肿/蛋白尿的症状。我们自己的实验室的工作表明，通过直接磷酸化的CX43功能丧失是血管舒张反应的立即丧失的基础，而其他人则显示了长期的细胞 - 细胞连接术进行了长期进行性分解，可能会导致进一步的水肿/蛋白质。值得注意的是，已知所有这些反应都是由对药物PP2敏感的SRC家族激酶介导的。前斜率的治疗很困难，部分原因是对疾病起源的了解有限，并且未能识别出单一一致的负责任内分泌因子。我们最近指出，在后受体水平上，许多潜在的负生长因子和细胞因子的关键收敛点是SRC的共同调节。有限的初步数据表明，PP2可以在体外挽救内皮细胞功能障碍，以响应多种配体支持该假设。现在，我们建议开发高吞吐量方法至1）验证我们的假设，即生长因子和细胞因子通过CX43和细胞 - 细胞连接水平的内皮功能障碍的分子共同调节是通过常见的SRC（PP2敏感）途径（以及2）筛选出一种适用于人类的新家族的preciants sake and sake for Human sce consection and sake sake sake sake sake sake sake sake sake sake sake sake sake sake。 AIM 2特别是根据初步实验，显示了SRC抑制剂10,12同工型中的天然食品共轭亚油酸（CLA），可以在我们的烤箱模型中挽救抑制Ca2+信号传导功能。我们进一步假设10,12和其他CLA同工型将在HUVEC中恢复Ca2+反应和单层完整性，否则会受到成长的因素和细胞因子的抑制。进步的主要局限性是迄今为止使用的CA2+成像方法是耗时的，因此对多种骑马和药物进行筛查是不切实际的。电细胞底物阻抗传感器（ECIS）系统实时测量细胞单层的电阻，并提供高分辨率的临时视图，以临时视图多韦尔格式的细胞 - 细胞连接分解。这是，提出的目标将为血管舒张功能和单层完整性水平上的内皮功能障碍的启动和恢复提供两管齐下的高吞吐量筛选方法。为此：特定的目标1筛选生长因子和细胞因子，可抑制HUVEC和单层连接完整性中CX43介导的Ca2+信号传导的能力。特定的AIM 2屏幕CLA异构体具有HUVEC中Ca2+信号传导和单层完整性函数的能力。