Dissecting gene dysregulation at the maternal-fetal interface in preeclampsia

描述（由申请人提供）：我们正在测试一个假设，即来自子痫前期（PE）患者胎盘主要滋养细胞（TB）亚群的全球基因表达谱分析将导致鉴定在该综合征中起重要作用的新分子。这种策略的推动力是发现PE始终与某些胎盘病理相关。细胞滋养细胞（CTB）侵袭的间质成分通常局限于浅表蜕膜。同样，受影响的螺旋小动脉的数量和它们被改变的程度限制了血管内侵犯。来自患者胎盘的合胞滋养细胞（STBs）也有明显的变化，如合胞结。这些改变伴随着分子变化，其中许多已通过PE中通常在结核病功能方面发挥重要作用的分子表达谱确定。然而，我们已经开始采用无偏见的方法来更全面地了解PE中TB基因表达的变化。这种方法承认我们对该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来分析各种严重PE （sPE）中的CTB基因表达。具体来说，从sPE患者和对照组女性的胎盘中分离的CTBs培养48小时，使其能够分化/侵袭。微阵列分析显示，spe相关的一组CTB基因上调，在培养期结束时，恢复到控制水平。它们包括先前与PE相关的因子和许多新分子，包括血管生成调节剂SEMA3B。我们继续表明，这种神经匹林-1和-2配体水平的升高，通过包括通过PI3K/AKT和GSK3通路下调VEGF信号传导在内的机制，表型化了PE对CTBs的许多影响。在sPE CTBs中也观察到相同的变化。我们的数据支持这样的理论：在PE中，体内环境损害CTB的分化/侵袭，差异表达的分子参与了机制，临床症状是由患者特异性因素决定的。在确定了sPE中侵袭性CTBs的遗传特征后，我们现在提出对其他三种受影响的TB人群，STBs，合胞结和血管内CTBs进行无偏倚分析。明确;我们将使用激光显微解剖从PE、sPE和对照胎盘中分离这些细胞，并使用全局转录谱方法来鉴定失调基因（目的1）。我们将采用我们的新细胞培养模型，人类结核祖细胞，来确定观察到的变化的功能意义（目的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。这些发现也可能具有重要的转化潜力，例如，失调分子的一个子集可能是循环生物标志物和/或改善胎盘功能的治疗靶点，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.