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，缺氧会导致胎儿生长减慢。迄今为止的研究未能找到一种方法，通过这种方法可以早期发现这些严重受损的妊娠，也没有揭示缺氧导致胎儿生长受限的具体机制。我们使用了一种独特的人类慢性（高原诱导）缺氧模型来表明，尽管母体动脉氧张力显著降低，但缺氧并不是胎儿生长受限的直接原因；给胎盘和胎儿的氧气输送不减少，胎儿的耗氧量不受影响。相反，是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，过量的胎盘葡萄糖消耗，减少了向胎儿的转移，是胎儿生长受限的第一步。代谢重编程，也被称为“保氧”似乎是潜在的原因，在这种现象中，缺氧通过缺氧诱导因子-1 （HIF-1）转录因子介导的改变，主动和可逆地抑制氧化代谢和氧气消耗。我们的体内数据支持代谢重编程对确保胎儿存活至关重要，但以牺牲生长为代价。它发生在导致胎儿生长受限的其他因素的手术之前。确定缺氧导致胎儿生长减少的途径，将允许在不可逆的胎儿损害之前开发诊断测试和改进治疗方法。作为我们继续研究的一部分，我们希望建立一种小鼠模型，在这种模型中，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识别和测试。在这项应用中，我们建议建立一个小鼠模型，用于诱导胎盘特异性HIF-1敲低。我们将以HIF-1为目标，因为它是迄今为止描述的所有代谢重编程机制的调节关系。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传递到囊胚的外层细胞层，而不是内部细胞团，导致胎盘转导而不影响胎儿。为了避免胚胎致死性（这使先前的HIF-1敲低研究变得复杂），我们将使用诱导载体，使我们能够在怀孕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)开发用于敲除小鼠HIF-1的慢病毒工具；(2)在体内进行测试

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.