Dissecting gene dysregulation at the maternal-fetal interface in preeclampsia

描述(申请人提供)：我们正在测试这样一种假设，即对先兆子痫(PE)患者胎盘主要滋养细胞(TB)亚群的全球基因表达谱将导致识别在这种综合征中发挥重要作用的新分子。这一策略的推动力是发现PE始终与某些胎盘病理有关。细胞滋养层细胞(CTB)侵袭的间质成分通常局限于浅层蜕膜。同样，血管内侵袭受限于受累的螺旋小动脉的数量和它们被修饰的程度。患者胎盘中的合体滋养层细胞(STB)也有明显的变化，如合体结节。这些变化伴随着分子变化，其中许多变化是通过分析PE中通常在结核病功能方面发挥重要作用的分子的表达来确定的。然而，我们已经开始应用无偏见的方法来更全面地了解PE中发生的结核病基因表达的变化。这种方法承认我们对这种综合征的胎盘成分的了解有限。最近，我们使用转录组学方法分析了CTB基因在各种严重形式的PE(SPE)中的表达。具体地说，从SPE患者和对照妇女的胎盘中分离出的CTB被培养48小时以允许分化/侵袭。微阵列分析显示，SPE相关的一套CTB基因上调，在培养阶段结束时，这些基因恢复到对照水平。它们包括以前与PE相关的因子和许多新的分子，包括血管生成调节因子SEMA3B。我们进一步证明，上调这种神经粘连蛋白-1和-2配体的水平可以通过包括通过PI3K/AKT和GSK3�/�途径下调血管内皮生长因子信号的机制来复制PE对CTB的许多影响。在SPE、CTB中也观察到同样的变化。我们的数据支持这样的理论，即在PE中，体内环境损害了CTB的分化/侵袭，差异表达的分子参与了这一机制，临床症状由患者特有的因素决定。在确定了SPE中侵袭性CTB的遗传特征后，我们现在建议对其他三个受影响的结核病群体--STB、合胞体结节和血管内CTB--进行无偏见的分析。具体地说，我们将使用激光显微解剖从PE、SPE和对照胎盘中分离出这些细胞，并使用全球转录图谱方法来识别失调基因(目标1)。我们将使用我们的新细胞培养模型--人类结核病前体细胞，来确定所观察到的变化的功能意义(目标2)。我们认为，这些实验将为PE胎盘缺陷的分子基础提供新的信息。这些发现还可能具有显著的翻译潜力，例如，调节失调的分子的子集可能是改善胎盘功能的循环生物标记物和/或治疗靶点，我们的数据表明这是可能的。

DESCRIPTION (provided by applicant): We are testing the hypothesis that global gene expression profiling of the major trophoblast (TB) subpopulations of placentas from preeclampsia (PE) patients will lead to the identification of novel molecules that play important roles in this syndrome. The impetus for this strategy is the finding that PE is consistently associated with certain placental pathologies. The interstitial component of cytotrophoblast (CTB) invasion is frequently restricted to the superficial decidua. Likewise, endovascular invasion is constrained in terms of the number of spiral arterioles that are involved and the extent to which they are modified. Syncytiotrophoblasts (STBs) from placentas of affected patients also have overt changes such as syncytial knots. These alterations are accompanied by molecular changes, many of which have been identified by profiling, in PE, the expression of molecules that normally play important roles in terms of TB functions. However, we have begun to apply unbiased approaches to gain a more comprehensive understanding of changes in TB gene expression that occur in PE. This approach acknowledges our limited understanding of the placental component of this syndrome. Recently, we used a transcriptomics approach to profile CTB gene expression in various severe forms of PE (sPE). Specifically, CTBs that were isolated from the placentas of sPE patients and control women were cultured for 48 h to allow differentiation/invasion. Microarray analyses revealed sPE-associated upregulation of a suite of CTB genes that, by the end of the culture period, returned to control levels. They included factors previously associated with PE and many novel molecules, including the angiogenic regulator, SEMA3B. We went on to show that elevating levels of this neuropilin-1 and -2 ligand phenocopied many of the effects of PE on CTBs by mechanisms that include downregulating VEGF signaling through the PI3K/AKT and GSK3�/� pathway. The same changes were observed in sPE CTBs. Our data support the theory that, in PE, the in vivo environment impairs CTB differentiation/invasion, the differentially expressed molecules contribute to the mechanisms, and that the clinical signs are determined by patient-specific factors. Having identified a genetic signature for invasive CTBs in sPE, we now propose an unbiased analysis of the three other affected TB populations, STBs, syncytial knots and endovascular CTBs. Specifically; we will use laser microdissection to isolate these cells from PE, sPE and control placentas and a global transcriptional profiling approach to identify the dysregulated genes (Aim 1). We will employ our new cell culture model, human TB progenitor cells, to determine the functional significance of the observed changes (Aim 2). We think that these experiments will yield new information about the molecular bases of placental defects in PE. The findings could also have significant translational potential, e.g., a subset of the dysregulated molecules could be circulating biomarkers and/or therapeutic targets for improving placental function, which our data suggest is possible.