Cellular Mechanisms of Amniotic Fluid Volume Regulation

羊水量调节的细胞机制

• 批准号: 8100191
• 负责人: CECILIA CHEUNG
• 金额: $ 27.26万
• 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/8100191
• 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)：正常的羊水量(AF)对于正常的胎儿发育和良好的围产期结局是必不可少的。然而，调节房颤量的机制以及将房颤量维持在生理范围内的因素还不是很清楚。目前的理解表明，房颤水和溶质通过胎膜转移到胎儿血管中，使胎盘表面血管形成，这是调节发生的途径。这种膜内(IM)吸收途径由一个主动的整体转运成分和一个被动的扩散成分组成。此外，房颤和羊膜中的刺激和抑制因子对激活过程也有调节作用。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。在这一应用中，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。这些研究将在体外绵羊羊膜细胞和体内慢性插管的绵羊胎儿中进行。在特定的目标1中，我们将确定溶质通过羊膜细胞运输的细胞途径，并检验房颤是一个通过小窝的囊泡跨细胞过程的假说。我们将探讨VEGF165作为空泡运输的刺激因子和VEGF165b作为空泡运输的抑制因子的作用，以及可溶性VEGF受体1(sVEGFR-1)在拮抗VEGF生物活性中的作用。特异性目标2将测定在正常、IM吸收率增加或降低的情况下羊膜和羊膜中VEGF165和VEGF165b的mRNA和蛋白水平。将确定VEGF165水平与sVEGFR-1的相关性。在特定的目标3中，我们将研究VEGF165通过诱导血管内皮生长因子受体2来启动c-Src信号通路，从而激活空泡内吞和跨细胞所需的caveelin-1和Dynamin-2下游的激活。其他信号蛋白包括蛋白激酶C和磷脂酰肌醇3-激酶的参与也将被探索。具体目的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，并确定它们在羊膜细胞被动和主动转运中的作用。在特定的目的5中，我们将评估在正常、增加或减少房颤的情况下，刺激物VEGF165和抑制物VEGF165b和sVEGFR-1对绵羊胚胎IM吸收率的调节作用。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。总体而言，这些研究将阐明房颤的跨细胞囊泡转运途径，并确定在羊膜细胞中调节这一途径的刺激和抑制调节因子。此外，将对调节这些运输事件的信号转导级联进行调查。这一发现将有助于更好地理解羊水量异常的病因。 公共卫生相关性：拟议的研究将对羊水量调节机制产生一个新的理解水平。这一知识在开发治疗因羊水量异常引起的妊娠并发症方面是至关重要的。这些研究最终将改善羊水过少和羊水过多的临床管理，从而减少围产期和新生儿的发病率。