Modeling preeclampsia in mice with inducible placenta-specific gene expression

描述（由申请人提供）：子痫前期是一种严重的妊娠并发症，对母体和胎儿均有影响，全球约14%的妊娠相关孕产妇死亡是由子痫前期引起的。然而，尽管它对公共卫生产生了巨大影响，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。基于人体临床研究和啮齿类动物实验研究，提示循环中可溶性类纤维样酪氨酸激酶1 （sFlt- 1）水平异常升高，通过其对血管内皮生长因子（VEGF）活性的拮抗，导致全体性内皮功能障碍和该病的临床表现。因此，我们认为VEGF治疗可以逆转由母体循环中高sFlt-1水平引起的子痫前期表型。然而，在初步研究中，我们令人惊讶地发现，怀孕小鼠体内过度表达VEGF会导致血压升高、血清sFlt-1和可溶性内啡肽（sEng）水平升高以及肾脏组织学改变，这与PE在人类和动物模型中的临床结果相似。VEGF已被证明在多种情况下刺激多种细胞类型和胎盘外植体培养中sFlt-1的产生。然而，胎盘VEGF在妊娠不同阶段的作用、胎盘生成sFlt-1对其局部活性的调控意义以及其在胎盘中调节sFlt-1生成的潜在作用尚未得到研究，这主要是由于缺乏合适的动物模型来测试VEGF对胎盘的特异性作用。我们假设胎盘中的VEGF活性在妊娠期间通过局部产生sFlt-1以胎盘发育阶段特异性的方式调节，胎盘中的VEGF水平可能是该组织中sFlt-1产生增加的主要刺激因素。本研究的目的是，首先，开发一种新颖的、可诱导的、胎盘特异性基因表达系统，通过活体成像技术监测整个妊娠期间的基因表达。该技术将基于已建立的病毒将基因特异性传递到胎盘组织的方法，有效控制基因表达水平的一致性，以及严格控制的诱导启动子，该启动子允许快速打开和关闭表达。然后，我们将使用该诱导系统检查妊娠期间不同时间点胎盘VEGF表达水平的影响。为了明确sFlt-1在妊娠不同阶段的生理作用，我们将利用靶向sFlt-1的短发夹RNA （shRNA）在胎盘中的特异性表达，选择性地敲低sFlt-1在胎盘中的表达。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，使用了一种新开发的、可诱导的、胎盘特异性基因调控系统，将极大地促进胎盘疾病和正常胎盘发育研究的新动物模型的发展，并将极大地促进妊娠并发症治疗的发展。

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.