Recent studies have shown that amniotic fluid (AF) volume is regulated by modulating the rate of intramembranous absorption and that vascular endothelial growth factor (VEGF) may play a pivotal role in this regulation. This absorption occurs against hydrostatic, concentration and experimentally induced osmotic gradients. However, the mechanisms mediating intramembranous transport as well as the regulation of intramembmnous absorption are unknown. The proposed studies utilize chronically catheterized fetal sheep to characterize intramembranous transport mechanisms and determine the underlying cellular and molecular regulation during normoxia and during 3 forms of fetal hypoxia which individually produce increased, unchanged, or reduced AF volume. We will determine the contribution of diffusion versus bulk transport to intramembranous absorption of water and solutes. These interpretations will be confirmed by measuring the intramembranous unidirectional fluxes of specific solutes in the AF to fetal and fetal to AF directions. The cellular and molecular mechanisms which regulate passive and bulk transfer through the intramembranous pathway will be determined by studying the effects of VEGF on transport in isolated amniotic membrane and amnion cells in culture. Our overall hypothesis is that intramembranous bulk flow occurs by unidirectional vesicular transport; that hypoxia augments bulk flow but not passive diffusion; and that the rate of vesicular transport is determined by the abundance and mobility of vesicles in the amnion. We further hypothesize that hypoxia augments VEGF expression which in turn regulates caveolin-1 expression and/or increases vesicle abundance and mobility. The proposed studies are important because oligohydramnios is a frequent clinical problem associated with fetal hypoxia accounting for many in utero fetal deaths due to cord compression and neonatal deaths due to respiratory distress. Polyhydramnios is associated with premature delivery and fetal diseases including anemic hypoxia. The proposed studies will lead to a dramatic improvement in our understanding of AF volume regulation under normal as well as hypoxic conditions. This knowledge will promote a rational basis for improved therapies to treat AF volume abnormalities, thereby helping to reduce fetal and neonatal morbidity and mortality.
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