Cellular Mechanisms of Amniotic Fluid Volume Regulation

羊水量调节的细胞机制

• 批准号: 7885966
• 负责人: CECILIA CHEUNG
• 金额: $ 27.55万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7885966
• 关键词: 1-Phosphatidylinositol 3-Kinase; AQP1 gene; Active Biological Transport; Address; Amniotic Fluid; Binding; Biological Process; Blood Vessels; Catheterization; Caveolae; Cell model; Cells; Chorion; Chronic; Clinical Management; Diffusion; Dynamin 2; Endocytosis; Epithelium; Etiology; Event; Experimental Models; Fetal Development; Fetal Membranes; Fetus; Gene Expression; Goals; In Vitro; Knowledge; Lead; Liquid substance; Mediating; Mediation; Membrane; Membrane Microdomains; Messenger RNA; Minor; Modeling; Molecular; Molecular Weight; Oligohydramnios; Outcome; Pathway interactions; Perinatal; Physiological; Placenta; Polyhydramnios; Pregnancy Complications; Process; Protein Isoforms; Protein Kinase; Protein Kinase C; Proteins; Regulation; Relative (related person); Role; SRC gene; Sheep; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Sister; Surface; Testing; Tissues; Transport Process; Urea; VEGF165; Vascular Endothelial Growth Factor Receptor; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors; Water; absorption; amnion; base; bevacizumab; caveolin 1; fetal; fetal blood; improved; in vivo; inhibitor/antagonist; insight; macromolecule; monolayer; neonatal morbidity; neutralizing antibody; novel; public health relevance; receptor; research study; response; solute; therapeutic development; therapy development; transcytosis; water channel

DESCRIPTION (provided by applicant): A normal volume of amniotic fluid (AF) is essential for normal fetal development with favorable perinatal outcome. However the mechanisms that regulate AF volume and the factors that maintain the volume within the physiological range are not well understood. The current understanding suggests that the transfer of AF water and solutes across the fetal membranes into fetal blood vessels that vascularize the surface of the placenta is the pathway where regulation occurs. This intramembranous (IM) pathway for AF absorption is constituted by an active bulk transport component and a passive diffusional component. In addition, the active process is regulated by stimulatory and inhibitory factors in the AF and amniotic membrane. Although the existence of these regulatory factors has been proposed, their identity and mechanisms of action are not known. In this application, we propose to elucidate the cellular pathways of IM transport and decipher the factors that regulate these pathways. These studies will be carried out in ovine amnion cells in vitro and chronically catheterized ovine fetuses in vivo. In Specific Aim 1, we will identify the cellular pathway for transport of solutes across amnion cells and test the hypothesis that AF transport is a vesicular transcytotic process via caveolae. We will investigate the role of VEGF165 as a stimulator and VEGF165b as an inhibitor of caveolar transport, as well as the effect of the soluble VEGF receptor 1 (sVEGFR-1) in antagonizing VEGF bioactivity. Specific Aim 2 will determine VEGF165 and VEGF165b mRNA and protein levels in amnion cells and amniotic membranes under conditions of normal, increased or decreased IM absorption rates. The correlation of VEGF165 levels with sVEGFR-1 will be determined. In Specific Aim 3, we will examine the VEGF165 activation of caveolar transcytosis by induction of VEGF receptor 2 to initiate a c-Src signaling pathway leading to downstream activation of cavoelin-1 and dynamin-2 as required for caveolar endocytosis and transcytosis. The involvement of other signaling proteins including protein kinase C and phosphatidylinositol 3-kinase will be explored. Specific Aim 4 will investigate the expression of the water channel proteins aquaporin 1, 3 and 9 in amnion cells and determine their effects on passive and active transport across amnion cells. In Specific Aim 5, we will evaluate the in vivo function of the stimulator VEGF165 and the inhibitors VEGF165b and sVEGFR-1 in modulating IM absorption rate in ovine fetuses under conditions of normal, increased or decreased AF volume. We anticipate the in vivo results to support the in vitro findings that VEGF165 is an important determinant of IM absorption and that its stimulatory effect is antagonized by VEGF165b and sVEGFR-1. Overall these studies will elucidate the transcellular vesicular pathway for AF transport and determine the stimulatory and inhibitory regulatory factors that modulate this pathway in amnion cells. Further, the signal transduction cascades that mediate these transport events will be investigated. The findings will lead to an improved understanding of the etiology of amniotic fluid volume abnormalities. PUBLIC HEALTH RELEVANCE: The proposed studies will generate a new level of understanding for the mechanisms of amniotic fluid volume regulation. This knowledge is crucial in the development of therapies for the treatment of pregnancy complications due to abnormalities in amniotic fluid volume. The studies will ultimately lead to improvement in the clinical management of oligohydramnios and polyhydramnios, thereby reducing perinatal and neonatal morbidity.

描述（由申请人提供）：正常量的羊水（AF）对于胎儿的正常发育和良好的围产期结局至关重要。然而，调节 AF 容量的机制以及将容量维持在生理范围内的因素尚不清楚。目前的理解表明，AF 水和溶质穿过胎膜进入胎儿血管，使胎盘表面血管化，这是发生调节的途径。这种 AF 吸收的膜内 (IM) 途径由主动大量转运成分和被动扩散成分构成。此外，活性过程受到 AF 和羊膜中的刺激和抑制因子的调节。尽管已经提出这些调节因子的存在，但它们的身份和作用机制尚不清楚。在此应用中，我们建议阐明 IM 运输的细胞途径并破译调节这些途径的因素。这些研究将在体外的绵羊羊膜细胞和体内长期插入导管的绵羊胎儿中进行。在具体目标 1 中，我们将确定跨羊膜细胞转运溶质的细胞途径，并检验 AF 转运是通过小凹的囊泡转胞吞过程的假设。我们将研究 VEGF165 作为刺激剂和 VEGF165b 作为细胞小穴转运抑制剂的作用，以及可溶性 VEGF 受体 1 (sVEGFR-1) 在拮抗 VEGF 生物活性中的作用。具体目标 2 将测定正常、增加或减少 IM 吸收率条件下羊膜细胞和羊膜中 VEGF165 和 VEGF165b mRNA 和蛋白质水平。将确定 VEGF165 水平与 sVEGFR-1 的相关性。在具体目标 3 中，我们将通过诱导 VEGF 受体 2 启动 c-Src 信号通路，从而根据小穴内吞作用和转胞吞作用所需的作用，导致下游的 cavoelin-1 和 dynamin-2 激活，从而检查 VEGF165 对小穴转胞吞作用的激活。将探讨其他信号蛋白（包括蛋白激酶 C 和磷脂酰肌醇 3-激酶）的参与。具体目标 4 将研究羊膜细胞中水通道蛋白水通道蛋白 1、3 和 9 的表达，并确定它们对羊膜细胞被动和主动运输的影响。在具体目标 5 中，我们将评估刺激剂 VEGF165 以及抑制剂 VEGF165b 和 sVEGFR-1 在正常、增加或减少 AF 体积的条件下调节绵羊胎儿 IM 吸收率的体内功能。我们预计体内结果将支持体外发现，即 VEGF165 是 IM 吸收的重要决定因素，并且其刺激作用被 VEGF165b 和 sVEGFR-1 拮抗。总体而言，这些研究将阐明 AF 转运的跨细胞囊泡途径，并确定在羊膜细胞中调节该途径的刺激和抑制调节因子。此外，还将研究介导这些运输事件的信号转导级联。这些发现将有助于更好地了解羊水量异常的病因。 公共健康相关性：拟议的研究将使人们对羊水量调节机制有了新的认识。这些知识对于开发治疗因羊水量异常引起的妊娠并发症的疗法至关重要。这些研究最终将改善羊水过少和羊水过多的临床管理，从而降低围产期和新生儿的发病率。