Mechanisms Modulating the Association between the ENG Pathway and Preeclampsia

描述(申请人提供)：先兆子痫(PE)的病理生理机制尚不清楚，也缺乏有效的策略/治疗方法来成功降低全球孕产妇和新生儿的发病率/死亡率。该项目的长期目标是提高我们对PE生物学基础的了解，以便开发有效的预防、检测和治疗干预措施，以识别处于危险中的妇女，并改善受PE影响的母亲和婴儿的健康状况。本项目旨在解决我们对PE病理生理学认识上的重大空白，重点在于提高我们对Enoglin(ENG)途径在PE中的异常调节的理解。我们知道，在临床表现为PE之前，循环中的可溶性Enoglin(Seng)蛋白和胎盘/血液中ENG mRNA的表达升高，而啮齿动物体内Seng水平的升高会产生一种PE样综合征，这种综合征可以被其他抗血管生成因子放大。我之前已经在美国高加索人和非裔美国人的样本中证明了ENG途径的基因变异与PE有关。然而，目前尚不清楚ENG途径中的遗传变异是否会影响ENG mRNA的表达和/或Seng水平，或者是否代谢因素(例如，PE中的维生素D下降)调节细胞Seng的输出。这个项目解决了endoglin途径失调的机制和对我们以前的遗传发现的验证，这两个都是必要的，以提供最终将这些发现转化为临床所需的证据基础。首先，使用候选基因病例对照设计，我们将检验这样一个假设，即我们的基因关联发现存在于比尔和梅林达·盖茨资助的全球怀孕共同实验室的其他人群中，该实验室是由&gt；20个研究小组组成的生物库联盟，研究不良妊娠结果。其次，我们将使用病例对照设计，对ENG进行重点基因分型(错义单核苷酸多态性评估；测序)，以确定与PE易感性相关的常见和/或罕见功能变异。第三，我们将解释ENG途径之间的潜在联系的机制：(A)测试妊娠血浆水平(例如，Seng)与ENG途径基因型(病例内或对照内设计)和妊娠结局的关系(病例对照设计)；(B)检验维生素D抑制在基础和刺激(低氧、PE血清)条件下培养的人滋养层细胞和子宫内皮细胞释放Seng的假设。方法为了实现这些特定目标，与国家护理研究所通过识别高危个体的易感基因来促进健康和预防疾病的使命密切相关，包括通过I-PLEX(R)Gold SNP分析或TaqMan(R)等位基因识别进行基因收集、基于毛细管的测序、利用ELISA法和Western Blot进行蛋白质测量，以及在不同处理条件下(例如，氧气含量、维生素D浓度、患者血清)进行细胞培养。最终，这个项目的结果将提高我们对PE的病理生理基础的理解，并可能有助于为有PE风险的妇女确定预防、预测和治疗/可修改的目标。

DESCRIPTION (provided by applicant): The pathophysiology of preeclampsia (PE) is unclear, and effective strategies/treatments to successfully reduce global maternal and neonatal morbidity/mortality are lacking. The long term objective of this project is to improve our understanding of the biologic underpinnings of PE so that effective prevention, detection, and treatment interventions can be developed to identify women at risk and improve health outcomes of moms and babies affected by PE. This project aims to address significant gaps in our understanding of PE pathophysiology by focusing on improving our understanding of endoglin (ENG) pathway dysregulation in PE. We know that elevations in circulating soluble endoglin (sENG) protein and placental/blood ENG mRNA expression precede clinical presentation of PE, and increased levels of sENG in rodents produce a PE-like syndrome that can be amplified by other anti-angiogenic factors. I have previously demonstrated that genetic variation in the ENG pathway is associated with PE in American Caucasian and African American samples. However, it is unknown if genetic variability in the ENG pathway impacts ENG mRNA expression and/or sENG levels, or if metabolic factors (e.g., Vitamin D down in PE) modulate cellular sENG output. This project addresses mechanisms of endoglin pathway dysregulation and validation of our previous genetic findings, both of which are necessary to provide the evidence based needed to eventually translate these findings clinically. First, using a candidate gene case-control design, we will test the hypothesis that our genetic association findings are present in other populations from the Bill & Melinda Gates funded Global Pregnancy CoLaboratory, a biobank consortium of >20 research groups studying adverse pregnancy outcomes. Second, we will conduct focused genotyping (missense single nucleotide polymorphism assessment; sequencing) of ENG to identify common and/or rare functional variants that are involved in PE susceptibility, using a case-control design. Third, we will explor mechanisms underlying associations between the ENG pathway: (a) test the relationship of pregnancy plasma levels (e.g., sENG) to ENG pathway genotypes (within-case OR within-control design) and pregnancy outcome (case-control design); (b) test the hypothesis that Vitamin D attenuates sENG release from human trophoblast and uterine endothelial cell lines cultured under basal and stimulated (hypoxia, PE serum) conditions. Methods to achieve these specific aims, germane to the National Institute of Nursing Research's mission to promote health and prevent disease through identification of susceptibility genes for at- risk individuals, includ genotype collection with either the i-PLEX(R) Gold SNP assay or TaqMan(R) allelic discrimination, capillary based sequencing, protein measurement with ELISA assays and Western Blot, and cell culture under varying treatment conditions (e.g., oxygen content, Vitamin D concentration, patient serum). Ultimately, results from this project will improve our understanding of the pathophysiologic underpinnings of PE and may help to identify preventive, predictive, and therapeutic/modifiable targets for women at risk for PE.