Role of Hypoxia in Regulating Stromal-Epithelial Communication during Pregnancy

当胚泡附着在子宫腔上皮上时开始着床， 渗透到下面的基质中以牢固地嵌入子宫内膜。虽然许多方面的 植入仍然不清楚，新出现证据表明， 子宫内膜上皮细胞和基质细胞对于成功建立妊娠至关重要。旁分泌 对介导这种串扰的信号仍然知之甚少。我们最近的研究表明， 转录因子，缺氧诱导因子2 α（HIF 2 α），在子宫内膜间质中选择性诱导 植入时腔上皮下的细胞。这一发现意义重大，因为 长期以来，人们都知道母体在着床期间的环境是缺氧的。解决 在植入期间母体适应缺氧的机制，我们产生了Hif 2 αd/d小鼠， 子宫内膜中条件性缺乏HIF 2 α。在这些小鼠中，胚泡与子宫紧密贴壁 上皮，但不能维持与其牢固的附着，导致植入失败。子宫内膜间质细胞 HIF 2 αd/d小鼠表现出代谢因子的下调，如乳酸和氨基酸的子集， 一组独特的Rab GTP酶，调节细胞外囊泡（EV）（包括微囊泡）的分泌 和外泌体。已知EV介导细胞间通讯的新机制。缺氧 促进某些细胞的EV分泌支持HIF 2 α驱动子宫内膜EV分泌的概念 基质细胞代表了一种关键的适应性反应， 置入在拟议的研究中，我们将采用Hif 2 αd/d小鼠模型来检验假设， HIF 2 α通过调节早孕缺氧条件下的一种新的旁分泌信号机制 控制母体组织中的EV运输，并指导关键信号分子的转移， 从基质到腔上皮细胞的代谢物，以影响植入期间的上皮功能。 我们提出了三个具体目标来检验这一假设。在目标1中，我们将研究HIF 2 α介导的 EV在子宫内膜间质细胞中的运输并鉴定EV中的驻留分子货物。在目标2中，我们 研究子宫内膜间质细胞分泌的EV和代谢因子对子宫内膜异位症的影响。 上皮细胞的功能。在目的3中，我们将研究HIF 2 α在调节细胞分泌中的作用， 人子宫内膜间质细胞的功能。一种多管齐下的方法，利用以下方面的组合： 在适应性反应中具有特定缺陷的动物模型（ii）使用小鼠的功能分析， 人原代子宫内膜细胞，和（iii）基因组和蛋白质组学分析，以了解细胞间 这将使我们能够为有关贩运人口的重要未决问题提供答案， 缺氧对控制早期子宫内膜功能间质-上皮通讯的影响 怀孕

Implantation is initiated when the blastocyst attaches to the uterine luminal epithelium and subsequently penetrates into the underlying stroma to firmly embed into the endometrium. While many aspects of implantation remain unclear, emerging evidence indicates that intercellular communication between endometrial epithelial and stromal cells is vital for successful establishment of pregnancy. The paracrine signals that mediate this crosstalk remain poorly understood. Our recent studies revealed that the transcription factor, hypoxia-­‐inducible factor 2 alpha (HIF2α), is induced selectively in endometrial stromal cells subjacent to the luminal epithelium at the time of implantation. This finding is significant because it is known for a long time that the maternal environment during implantation is hypoxic. To address the mechanisms of maternal adaptation to hypoxia during implantation, we generated Hif2αd/d mice that conditionally lack HIF2α in the endometrium. In these mice, the blastocysts are closely apposed to the uterine epithelium but fail to sustain firm attachment to it, resulting in implantation failure. The uterine stromal cells of Hif2αd/d mice exhibited downregulation of metabolic factors, such as lactate and a subset of amino acids, and a distinct set of Rab GTPases that regulate secretion of extracellular vesicles (EVs), including microvesicles and exosomes. The EVs are known to mediate a novel mechanism of cell-cell communication. That hypoxia promotes EV secretion by certain cells support the concept that HIF2α-driven EV secretion by the endometrial stromal cells represents a critical adaptive response that promotes intercellular communication during implantation. In the proposed study, we will employ the Hif2αd/d mouse model to test the hypothesis that HIF2α regulates a novel paracrine signaling mechanism under the hypoxic conditions of early pregnancy by controlling EV trafficking in the maternal tissue and that it directs transfer of key signaling molecules and metabolites from the stromal to luminal epithelial cells to influence epithelial functionality during implantation. We have proposed three specific aims to test this hypothesis. In Aim 1, we will investigate HIF2α-mediated EV trafficking in endometrial stromal cells and identify the resident molecular cargo in EVs. In Aim 2, we will investigate the effects of EVs and metabolic factors secreted from endometrial stromal cells on the functionality of epithelial cells. In Aim 3, we will investigate the role of HIF2α in regulating the secretory function of human endometrial stromal cells. A multi-pronged approach, utilizing a combination of (i) a unique animal model harboring a specific defect in adaptive response (ii) functional analyses using mouse and human primary endometrial cells, and (iii) genomic and proteomic analyses to understand cell-to-cell trafficking mechanisms, will enable us to provide answers to important unresolved questions regarding the impact of hypoxia on stromal-epithelial communication controlling endometrial function during early pregnancy.