Cellular Mechanisms of Amniotic Fluid Volume Regulation

羊水量调节的细胞机制

• 批准号: 8477061
• 负责人: CECILIA CHEUNG
• 金额: $ 26.2万
• 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/8477061
• 关键词: 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; Phosphatidylinositols; 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; passive transport; 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.

描述（由申请人提供）：正常羊水量（AF）对于胎儿正常发育和良好的围产期结局是必不可少的。然而，调控心房颤动体积的机制和维持心房颤动体积在生理范围内的因素尚不清楚。目前的理解表明，心房颤动水和溶质通过胎膜进入胎盘表面血管的胎儿血管是发生调节的途径。这种AF的膜内吸收途径是由主动的大块运输成分和被动的扩散成分组成的。此外，活性过程受AF和羊膜的刺激和抑制因子的调节。虽然这些调节因子的存在已被提出，但它们的身份和作用机制尚不清楚。在这个应用中，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。这些研究将在体外羊羊膜细胞和体内长期插管的羊胎儿中进行。在Specific Aim 1中，我们将确定溶质在羊膜细胞间运输的细胞途径，并验证AF运输是一个通过小泡的囊泡胞吞过程的假设。我们将研究VEGF165作为刺激剂和VEGF165b作为囊泡运输抑制剂的作用，以及可溶性VEGF受体1 （sVEGFR-1）在拮抗VEGF生物活性中的作用。特异性Aim 2将测定羊膜细胞和羊膜中正常、增加或减少IM吸收率条件下VEGF165和VEGF165b mRNA和蛋白水平。将确定VEGF165水平与sVEGFR-1的相关性。在Specific Aim 3中，我们将通过诱导VEGF受体2启动c-Src信号通路，导致cavelin -1和dynamin-2的下游激活，从而研究VEGF - 165对腔泡胞吞作用的激活，这是腔泡内吞和胞吞作用所必需的。其他信号蛋白包括蛋白激酶C和磷脂酰肌醇3-激酶的参与将被探讨。特异性Aim 4将研究水通道蛋白水通道蛋白1、3和9在羊膜细胞中的表达，并确定它们对羊膜细胞被动和主动运输的影响。在特异性目标5中，我们将评估刺激剂VEGF165和抑制剂VEGF165b和sVEGFR-1在正常、AF体积增加或减少的情况下调节羊胎儿IM吸收率的体内功能。我们预计体内结果将支持体外研究结果，即VEGF165是IM吸收的重要决定因素，其刺激作用可被VEGF165b和sVEGFR-1拮抗。总的来说，这些研究将阐明AF转运的跨细胞囊泡途径，并确定羊膜细胞中调节这一途径的刺激和抑制调节因子。此外，介导这些转运事件的信号转导级联将被研究。研究结果将导致对羊水容量异常的病因学的进一步了解。