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）：正常体积的羊水（AF）对于正常的胎儿发育至关重要，并具有良好的围产期结局。但是，调节AF量的机制以及在生理范围内维持体积的因素尚不清楚。当前的理解表明，AF水和溶液在整个胎儿膜中的转移到胎儿血管中，使胎盘表面的血管是发生调节的途径。这种用于AF吸收的膜内（IM）途径是由主动散装转运成分和被动扩散成分组成的。此外，主动过程受AF和羊膜中的刺激和抑制因子调节。尽管已经提出了这些调节因素的存在，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转运的细胞途径并破译调节这些途径的因素。这些研究将在体内体外和慢性导管卵胎胎儿在卵巢羊膜细胞中进行。在特定的目标1中，我们将确定溶质跨羊膜细胞运输的细胞途径，并测试假设AF转运是通过小窝的囊泡经胞胞细胞过程。我们将研究VEGF165作为刺激剂和VEGF165B作为Caveolar转运的抑制剂的作用，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将在正常，增加或降低IM吸收率的条件下确定羊膜细胞和羊膜膜中的VEGF165和VEGF165B mRNA和蛋白质水平。将确定VEGF165水平与SVEGFR-1的相关性。在特定的目标3中，我们将通过诱导VEGF受体2诱导Caveolar transytytosis的VEGF165激活，以启动C-SRC信号传导途径，从而导致Cavealal-1和Dynamin-2的下游激活，这是Caveolar内吞作用和跨胞菌病的必需品。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将研究水通道蛋白在羊膜细胞中水通道蛋白1、3和9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。在特定的目标5中，我们将在正常，增加或减少AF量的条件下，在调节卵巢胎儿的IM吸收率中评估刺激剂VEGF165和抑制剂VEGF165B和SVEGFR-1的体内功能。我们预计，体内结果支持体外发现，即VEGF165是IM吸收的重要决定因素，并且其刺激作用与VEGF165B和SVEGFR-1拮抗。总的来说，这些研究将阐明用于AF转运的跨细胞囊泡途径，并确定调节羊膜细胞中该途径的刺激性和抑制性调节因子。此外，将研究介导这些运输事件的信号转导级联反应。这些发现将导致人们对羊水体积异常的病因的理解。 公共卫生相关性：拟议的研究将对羊水体积调节的机制产生新的了解。由于羊水量异常，这种知识对于治疗妊娠并发症的疗法至关重要。这些研究最终将改善少聚糖和多氢化物的临床管理，从而降低围产期和新生儿发病率。