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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD061541-04
Application #
8477061
Study Section
Pregnancy and Neonatology Study Section (PN)
Program Officer
Ilekis, John V
Project Start
2010-07-01
Project End
2015-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
4
Fiscal Year
2013
Total Cost
$262,011
Indirect Cost
$91,874
Name
Oregon Health and Science University
Department
Obstetrics & Gynecology
Type
Schools of Medicine
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
Brace, Robert A; Cheung, Cecilia Y; Anderson, Debra F (2014) Inhibitor of intramembranous absorption in ovine amniotic fluid. Am J Physiol Regul Integr Comp Physiol 306:R185-9
Brace, Robert A; Anderson, Debra F; Cheung, Cecilia Y (2014) Regulation of amniotic fluid volume: mathematical model based on intramembranous transport mechanisms. Am J Physiol Regul Integr Comp Physiol 307:R1260-73
Cheung, Cecilia Y; Beardall, Michael K; Anderson, Debra F et al. (2014) Prostaglandin E2 regulation of amnion cell vascular endothelial growth factor expression: relationship with intramembranous absorption rate in fetal sheep. Am J Physiol Regul Integr Comp Physiol 307:R354-60
Anderson, Debra F; Jonker, Sonnet S; Louey, Samantha et al. (2013) Regulation of intramembranous absorption and amniotic fluid volume by constituents in fetal sheep urine. Am J Physiol Regul Integr Comp Physiol 305:R506-11
Brace, Robert A; Anderson, Debra F; Cheung, Cecilia Y (2013) Fetal swallowing as a protective mechanism against oligohydramnios and polyhydramnios in late gestation sheep. Reprod Sci 20:326-30