Cellular Mechanisms of Amniotic Fluid Volume Regulation

羊水量调节的细胞机制

• 批准号: 8282758
• 负责人: CECILIA CHEUNG
• 金额: $ 27.61万
• 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/8282758
• 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. 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）对胎儿正常发育和良好的围产期结局至关重要。然而，调节AF体积的机制和将体积维持在生理范围内的因素还没有很好地理解。目前的理解表明，AF水和溶质穿过胎膜转移到使胎盘表面血管化的胎儿血管中是发生调节的途径。AF吸收的膜内（IM）途径由主动大量转运组分和被动扩散组分构成。此外，主动过程由AF和羊膜中的刺激和抑制因子调节。虽然已经提出了这些调节因子的存在，但它们的身份和作用机制尚不清楚。在这个应用程序中，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。这些研究将在体外绵羊羊膜细胞和体内长期插管的绵羊胎仔中进行。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转运是通过小窝的囊泡胞吞过程的假设。我们将研究VEGF 165作为刺激剂和VEGF 165 b作为小窝转运抑制剂的作用，以及可溶性VEGF受体1（sVEGFR-1）拮抗VEGF生物活性的作用。特异性目的2将在IM吸收率正常、增加或降低的条件下测定羊膜细胞和羊膜中的VEGF 165和VEGF 165 b mRNA和蛋白水平。将确定VEGF 165水平与sVEGFR-1的相关性。在特定目标3中，我们将通过诱导VEGF受体2启动c-Src信号传导途径，导致小窝内吞和胞吞转运所需的小窝蛋白-1和发动蛋白-2的下游激活，来检查VEGF 165对小窝胞吞转运的激活。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。具体目标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，并确定它们对跨羊膜细胞的被动和主动转运的影响。在特定目标5中，我们将评价刺激剂VEGF 165和抑制剂VEGF 165 b和sVEGFR-1在正常、增加或减少AF体积条件下调节绵羊胎儿IM吸收率的体内功能。我们预期体内结果支持体外研究结果，即VEGF 165是IM吸收的重要决定因素，其刺激作用可被VEGF 165 b和sVEGFR-1拮抗。总之，这些研究将阐明AF运输的跨细胞囊泡途径，并确定在羊膜细胞中调节该途径的刺激性和抑制性调节因子。此外，介导这些运输事件的信号转导级联将被调查。这一发现将有助于提高对羊水量异常病因的认识。 公共卫生相关性：拟议的研究将对羊水量调节机制产生新的理解水平。这方面的知识是至关重要的治疗方法的发展，用于治疗妊娠并发症，由于异常的羊水量。这些研究将最终导致羊水过少和羊水过多临床管理的改善，从而降低围产期和新生儿发病率。