Mechanisms Modulating the Association between the ENG Pathway and Preeclampsia

描述（由申请人提供）：先兆子痫（PE）的病理生理学尚不清楚，缺乏有效的策略/治疗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体育运动生物学基础的理解，以便制定有效的预防、检测和治疗干预措施，以确定有风险的妇女，并改善受体育运动影响的母亲和婴儿的健康状况。本项目旨在通过提高我们对内啡肽（ENG）通路失调的理解来解决我们对PE病理生理学理解的重大空白。我们知道循环可溶性内啡肽（sENG）蛋白和胎盘/血液ENG mRNA表达的升高先于PE的临床表现，并且啮齿动物中sENG水平的升高会产生PE样综合征，该综合征可被其他抗血管生成因子放大。我之前已经证明，在美国白种人和非裔美国人样本中，ENG途径的遗传变异与PE有关。然而，目前尚不清楚ENG通路的遗传变异是否会影响ENG mRNA表达和/或sENG水平，或者代谢因素（如PE中的维生素D下降）是否会调节细胞的sENG输出。该项目解决了内啡肽通路失调的机制，并验证了我们之前的遗传发现，这两者都是为最终将这些发现转化为临床所需的证据所必需的。首先，使用候选基因病例对照设计，我们将检验我们的遗传关联发现是否存在于比尔和梅琳达·盖茨资助的全球妊娠实验室的其他人群中，该实验室是一个由bbbb20个研究小组组成的生物库联盟，研究不良妊娠结局。其次，我们将采用病例对照设计，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，我们将探索ENG通路之间的潜在关联机制：(a)测试妊娠血浆水平（如sENG）与ENG通路基因型（病例内或对照内设计）和妊娠结局（病例对照设计）的关系；(b)验证在基础和刺激（缺氧，PE血清）条件下培养的人滋养细胞和子宫内皮细胞系，维生素D能减弱sENG释放的假设。实现这些特定目标的方法与美国国家护理研究所（National Institute of Nursing Research）通过识别高危人群的易感基因来促进健康和预防疾病的使命密切相关，包括使用i-PLEX(R) Gold SNP测定法或TaqMan(R)等位基因鉴别法收集基因型、基于毛细管测序、使用ELISA测定和Western Blot测定蛋白质、以及在不同处理条件下(例如氧含量、维生素D浓度，患者血清)。最终，该项目的结果将提高我们对PE病理生理基础的理解，并可能有助于确定PE风险女性的预防、预测和治疗/可修改目标。

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.