A murine model for placental metabolic reprogramming

描述（由申请人提供）：在主要病理条件下，如宫内生长受限（IUGR）和先兆子痫，缺氧导致胎儿生长减慢。到目前为止，研究未能开发出一种方法，使这些严重受损的怀孕可以早期检测，也没有揭示缺氧导致胎儿生长受限的具体机制。我们已经使用了一个独特的人类慢性（海拔诱导）缺氧模型，以表明，尽管母体动脉血氧分压大幅下降，缺氧是不是胎儿生长受限的近因;氧气输送到胎盘和胎儿不减少，胎儿耗氧量不受影响。相反，胎儿循环葡萄糖浓度、胎儿葡萄糖消耗和胎儿胰岛素水平显著降低。这些体内研究结果表明，胎盘葡萄糖消耗过多，减少了向胎儿的转移，是胎儿生长受限的起始步骤。代谢重编程，也称为“氧气节约”，似乎是根本原因，缺氧通过缺氧诱导因子-1（HIF-1）转录因子介导的改变主动且可逆地抑制氧化代谢和氧气消耗的现象。我们的体内数据支持代谢重编程对于确保胎儿存活至关重要，但以生长为代价。它发生在其他因素导致胎儿生长受限的手术之前。鉴定缺氧引起胎儿生长减少的方法将允许在不可逆的胎儿损害之前开发诊断测试和改善治疗。作为我们继续研究的一部分，我们希望开发一种小鼠模型，其中胎盘代谢重编程机制及其对胎儿胎盘生长的影响可以在体内鉴定和测试。在本申请中，我们建议开发一种诱导型胎盘特异性HIF-1敲低的小鼠模型。我们将靶向HIF-1，因为它是迄今为止描述的所有代谢重编程机制的调节联系。为了限制对胎盘的敲除，我们将利用最近描述的慢病毒转导技术将HIF-1 <$shRNAmir递送到胚泡的外细胞层而不是内细胞团，从而导致胎盘转导而不影响胎儿。为了避免胚胎致死性，这使以前的（全身性）HIF-1敲低研究复杂化，我们将使用诱导型载体，使我们能够在妊娠中的生理相关时间点抑制胎盘HIF-1缺氧反应，一旦主要结构发育完成。我们将通过以下目标开发该模型：（1）开发用于敲低小鼠HIF-1的慢病毒工具和（2）测试体内HIF-1的表达。 使用HIF-1 shRNAmir的慢病毒转导的HIF-1胎盘敲低模型。该模型的建立将对胎儿缺氧和生长发育的研究产生重要影响。 公共卫生相关性：氧气供应的减少有助于减少胎儿生长的主要问题，如宫内生长受限和先兆子痫。然而，我们在人类中的研究结果表明，在氧气减少的情况下，胎盘改变了转移到胎儿的营养物质的混合，以减少生长为代价维持胎儿的存活，开始了胎儿生长限制的过程。作为我们持续研究的一部分，我们建议 建立一个小鼠模型，在这个模型中，我们可以在怀孕的不同时间点改变胎盘对氧气使用的调节，以研究营养供应的改变对胎儿生长的影响。

DESCRIPTION (provided by applicant): Hypoxia contributes to reduced fetal growth in major pathological conditions such as intrauterine growth restriction (IUGR) and preeclampsia. Research thus far has failed to develop a means by which these severely compromised pregnancies can be detected early nor has it revealed the specific mechanisms by which hypoxia leads to fetal growth restriction. We have used a unique human model of chronic (altitude-induced) hypoxia to show that despite a substantial decrement in maternal arterial oxygen tension, hypoxia is not the proximate cause of the fetal growth restriction; oxygen delivery to the placenta and fetus is not reduced and fetal oxygen consumption is unaffected. Instead, it is fetal circulating glucose concentrations, fetal glucose consumption and fetal insuli levels that are significantly reduced. These in vivo findings point to excess placental glucose consumption, reducing transfer to the fetus, as an initiating step in fetal growth restriction. Metabolic reprogramming, also known as "oxygen sparing" appears to be the underlying cause, a phenomenon in which hypoxia actively and reversibly inhibits oxidative metabolism and oxygen consumption through alterations mediated by the Hypoxia-Inducible Factor-1 (HIF-1) transcription factor. Our in vivo data supports that metabolic reprogramming is crucial for ensuring fetal survival, but at the expense of growth. It occurs prior to the operation of the othe factors contributing to fetal growth restriction. Identification of the means by which hypoxia initiates reduction in fetal growth will permit development of diagnostic tests and ameliorative therapies for use, prior to irreversible fetal compromise. As a part of our continuing studies we wish to develop a murine model in which placental metabolic reprogramming mechanisms and the resultant effects on fetoplacental growth can be identified and tested in vivo. In this application we propose to develop a murine model for inducible, placenta-specific HIF-1 knockdown. We will target HIF-1 since it is the regulatory nexus for all metabolic reprogramming mechanisms described thus far. To restrict knockdown to the placenta, we will take advantage of a recently described technique for lentiviral transduction to deliver HIF-1¿shRNAmir to the outer cell layer of the blastocyst but not the inner cell mass, leading to placental transduction without effects on the fetus. To avoid the embryonic lethality which has complicated previous (systemic) HIF-1 knockdown studies, we will use an inducible vector, allowing us to inhibit placental HIF-1 hypoxic responses at physiologically relevant time points in pregnancy, once major structural development is complete. We will develop this model through the following aims: (1) developing the lentiviral tools for knock down of murine HIF-1 and (2) testing an in vivo model for placental knockdown of HIF-1 using lentiviral transduction of HIF-1 shRNAmir. Development of this model will have a significant impact on studies of fetal hypoxia and growth. PUBLIC HEALTH RELEVANCE: Reduced availability of oxygen contributes to reduced fetal growth in major problems such as intrauterine growth restriction and preeclampsia. Our results in the human however suggest that in condition of reduced oxygen, the placenta alters the mix of nutrients transferred to the fetus, maintaining fetal survival at the cost of reduced growth, an starting the process of fetal growth restriction. As a part of our continuing studies we propose to generate a mouse model in which we can modify the placental regulation of oxygen use at different points in pregnancy to investigate the effects of alteration in nutrient supply on fetal growth.