Modeling preeclampsia in mice with inducible placenta-specific gene expression

描述（由申请人提供）：先兆子痫是一种严重的妊娠并发症，影响母亲和胎儿，估计占全球妊娠相关孕产妇死亡的14%。然而，尽管其对公共卫生的影响很大，但其原因仍然知之甚少，并且没有已知的治疗方法可以有效地减轻母亲和胎儿的风险。基于在人类中的临床研究和在啮齿动物中的实验研究，已经表明可溶性fms样酪氨酸激酶1（sFlt- 1）的循环水平的异常升高通过其对血管内皮生长因子（VEGF）活性的拮抗作用而促成全身性内皮功能障碍和疾病的临床表现。因此，认为VEGF治疗可以逆转由母体循环中高sFlt-1水平引起的先兆子痫表型。然而，在初步的研究中，我们发现，令人惊讶的是，VEGF在怀孕小鼠中的过度表达导致血压升高，sFlt-1和可溶性内皮糖蛋白（sEng）的血清水平增加，以及肾脏的组织学变化，与人类和动物模型中PE的临床结果相似。已显示VEGF在多种情况下刺激几种细胞类型和胎盘外植体培养物中的sFlt-1产生。然而，胎盘VEGF在妊娠不同阶段的作用、通过胎盘产生sFlt-1调节其局部活性的意义以及其在调节胎盘中sFlt-1产生中的潜在作用从未被研究过，主要是由于缺乏合适的动物模型来测试VEGF的胎盘特异性作用。我们假设胎盘中的VEGF活性在妊娠期间通过sFlt-1的局部产生以胎盘发育阶段特异性方式调节，并且胎盘VEGF水平可能是该组织中sFlt-1产生增加的主要刺激。在这个建议中的研究设计，首先，开发一种新的，可诱导的，胎盘特异性的基因表达系统，具有监测整个怀孕过程中的基因表达的能力，活在体内成像。该技术将基于已建立的方法，用于将基因特异性地病毒递送至胎盘组织，有效控制基因表达水平的一致性，以及严格控制的诱导型启动子，其允许表达快速地开启和关闭。然后，我们将研究在不同的时间点，在怀孕期间使用这种诱导系统的胎盘VEGF表达的不同水平的影响。为了确定sFlt-1在妊娠不同阶段的生理作用，我们将使用靶向sFlt-1的短发夹RNA（shRNA）的胎盘特异性表达来选择性地敲低胎盘中的sFlt-1表达。这些实验的结果将首次描述VEGF和sFlt-1在胎盘中调节VEGF活性的局部相互作用。此外，这种全新的方法，使用新开发的，可诱导的，胎盘特异性基因调控系统，将大大促进新的动物模型的开发，用于研究胎盘疾病和正常胎盘发育，并将大大促进妊娠并发症的治疗方法的开发。 公共卫生相关性：这项工作解决了妊娠并发症先兆子痫的根本原因，先兆子痫影响全世界5-7%的妊娠，是孕产妇和胎儿发病率和死亡率的主要原因。它开发了一种新的方法来研究不同因素在胎盘功能和疾病中的作用，使用小鼠作为模型系统，特别是解决可溶性fms样酪氨酸激酶1（sFlt-1）的生理作用和调节机制，可溶性fms样酪氨酸激酶1（sFlt-1）是血管内皮生长因子受体1（Flt-1）的可溶形式，其在患有先兆子痫的妇女的血液中以高水平存在，并且被认为是引起这种疾病的母体症状的原因。了解这种蛋白质的正常和异常功能将对临床治疗的设计产生直接和直接的影响，以保护母亲免受先兆子痫的不良后果，并将提供关键的理解如何设计这些治疗方法，以进一步保护胎儿。

DESCRIPTION (provided by applicant): Preeclampsia is a serious complication of pregnancy, affecting both the mother and fetus and causing an estimated 14% of pregnancy-related maternal deaths worldwide. Despite its large public health impact, however, its causes are still poorly understood, and there are no known treatments that effectively alleviate the risks for both mother and fetus. Based on clinical studies in humans and experimental studies in rodents, it has been suggested that the abnormal elevation of the circulating levels of soluble fms-like tyrosine kinase 1 (sFlt- 1) contributes to the systemic endothelial dysfunction and clinical manifestations of the disease through its antagonism of vascular endothelial growth factor (VEGF) activity. As a result, it is believed that VEGF treatment may reverse the preeclamptic phenotype caused by high sFlt-1 levels in the maternal circulation. However, in preliminary studies, we found, surprisingly, that overexpression of VEGF in pregnant mice results in elevated blood pressure, increased serum levels of sFlt-1 and soluble endoglin (sEng), and histological changes in the kidneys, similar to the clinical findings of PE in humans and animal models. VEGF has been shown to stimulate sFlt-1 production in several cell types and placental explant cultures in multiple contexts. However, the roles of placental VEGF at different stages of pregnancy, the significance of regulation of its local activity by placental production of sFlt-1, and its potential role in regulating sFlt-1 production in the placenta have never been examined, primarily due to lack of suitable animal models to test the placenta-specific effects of VEGF. We hypothesize that VEGF activity in the placenta is regulated in a placental developmental stage- specific manner during pregnancy through local production of sFlt-1, and placental VEGF levels may be a primary stimulus for increased production of sFlt-1 in this tissue. Studies in this proposal are designed, first, to develop a novel, inducible, placenta-specific gene expression system, with the ability to monitor gene expression throughout pregancy by live in vivo imaging. This technique will be based on established methods for viral delivery of genes specifically to placental tissue, effective control of consistency in gene expression levels, and a tightly controlled inducible promoter which allows expression to be rapidly switched both on and off. We will then examine the effects of different levels of placental VEGF expression at various time points during pregnancy using this inducible system. To define the physiological role of sFlt-1 during different stages of pregnancy, we will selectively knock down sFlt-1 expression in the placenta using placenta-specific expression of a short hairpin RNA (shRNA) targeted against sFlt-1. The results of these experiments will delineate, for the first time, the local interactions of VEGF and sFlt-1 in regulating VEGF activity in the placenta. Additionally, this entirely novel approach, using a newly-developed, inducible, placenta-specific gene regulation system, will greatly facilitate development of new animal models for the study of both placental disease and normal placental development and will greatly enable the development of therapies for complications of pregnancy. PUBLIC HEALTH RELEVANCE: This work addresses the fundamental causes of the pregnancy complication preeclampsia, which affects 5-7% of pregnancies worldwide and is a leading cause of maternal and fetal morbidity and mortality. It develops a new method to study the roles of different factors in placental functions and diseases, using mice as a model system, specifically addressing the physiological role and mechanism of regulation of soluble fms-like tyrosine kinase 1 (sFlt-1), a soluble form of the vascular endothelial growth factor receptor 1 (Flt-1), which is found at high levels in the blood of women with preeclampsia and is believed to cause the maternal symptoms in this disease. Understanding the normal and abnormal functions of this protein will have a direct and immediate impact on the design of clinical therapies for protecting the mother from the adverse consequences of preeclampsia and will provide critical understanding of how these therapies can be designed to further protect the fetus.