Modeling preeclampsia in mice with inducible placenta-specific gene expression

先兆子痫是妊娠的一种严重并发症，影响母亲和胎儿，并导致 据估计，全世界与怀孕有关的孕产妇死亡人数中有14%。尽管它对公共健康有很大的影响， 然而，其原因仍然知之甚少，也没有已知的治疗方法来有效地缓解 对母亲和胎儿都有风险。基于人类的临床研究和啮齿动物的实验研究，它 已有研究表明循环中可溶性FMS样酪氨酸激酶1水平的异常升高 (sflt-1)通过以下途径参与全身内皮功能障碍和疾病的临床表现 其拮抗血管内皮生长因子(VEGF)活性。因此，人们认为，血管内皮生长因子 治疗可能逆转由母体循环中高水平的sFlt-1引起的先兆子痫的表型。 然而，在初步研究中，我们令人惊讶地发现，在怀孕小鼠中过度表达血管内皮生长因子会导致 在高血压和血清sFlt-1和sEnoglin(Seng)水平升高以及组织学变化中 肾脏，类似于PE在人类和动物模型中的临床发现。血管内皮细胞生长因子已被证明 在多种情况下刺激几种细胞类型和胎盘组织培养中sflt-1的产生。然而， 胎盘血管内皮生长因子在妊娠不同阶段的作用及其局部活性调节的意义 通过胎盘产生sflt-1，以及它在胎盘中调节sflt-1产生的潜在作用，从未 这主要是由于缺乏合适的动物模型来测试血管内皮生长因子对胎盘的特定影响。 我们假设胎盘中的血管内皮生长因子活性在胎盘发育阶段中受到调节。 妊娠期通过局部产生sFlt-1的方式，可能与胎盘血管内皮生长因子水平有关 刺激增加sFlt-1在该组织中的产生。这项建议中的研究首先是为了开发 一种新型的、可诱导的、具有监测基因表达能力的胎盘特异性基因表达系统 在整个妊娠过程中，通过活体成像。这项技术将基于现有的病毒治疗方法 将基因特异性地输送到胎盘组织，有效控制基因表达水平的一致性，以及 一种严格控制的可诱导启动子，允许表达同时快速开启和关闭。我们会 然后检测不同水平的胎盘血管内皮生长因子在不同时间点的表达 使用这种可诱导系统怀孕。明确sFlt-1在高血压不同阶段的生理作用 妊娠期间，我们将利用胎盘特异性表达选择性地下调sFlt-1在胎盘中的表达 针对sFlt-1的短发夹状RNA(ShRNA)。这些实验的结果将描绘出，对于 首次研究了血管内皮生长因子和sFlt-1在调节胎盘血管内皮生长因子活性中的局部相互作用。此外，这一点 全新的方法，使用新开发的，可诱导的，胎盘特异的基因调控系统，将 极大地促进了研究胎盘疾病和正常的新动物模型的发展 胎盘发育，并将极大地促进妊娠并发症治疗的发展。

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 the human 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 and 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, has 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.