Normal pregnancy is associated with dramatic increases in utero- and feto- placental blood flows which are directly correlated with fetal growth as well as neonatal birth weights and survivability. These increases in placental blood flows during late gestation result from both vasodilation and neovascularization. During the third trimester of ovine pregnancy, we and others reported expression of the potent angiogenic factors basic Fibroblast Growth Factor (bFGF) and Vascular Endothelial Growth Factor(VEGF) in ovine placental tissues. We also noted that the maternal and fetal compartments are associated with very pronounced productions of angiotensin II (ANG II); e.g. in sheep, fetal ANG II production is approximately 10-fold greater than maternal production. Overall hypothesis; Expression of the placental angiogenic factors, bFGF and VEGF, is modulated by ANG II via its AT-1 and/or AT-2 receptor subtypes and that these two growth factors modulate placental vascular growth to control blood flow. We will address this by using explant cultures of ovine placentomes and ovine fetoplacental arterial endothelial cell lines from the third trimester of ovine pregnancy. We propose to determine in the fetal vs maternal component of the placentome four specific aims; 1) if bFGF and VEGF expression and angiogenesis are increased during the third trimester; 2) if ANG II receptors AT-1 and/or AT-2 subtype binding/expression increase at this time; 3) if exogenous ANG II increases the expression of bFGF and VEGF, whether this is mediated through the AT-1 and/or the AT-2 receptor, and whether this is reflected in increases in angiogenic activity; and 4) if ANG II, via AT-1 and/or AT-2 receptors, directly affect the angiogenic and growth factor changes in placental artery endothelial cell lines. Expression of bFGF and VEGF will be evaluated functionally using techniques of immunoneutralization of explant- conditioned media and determining its effects on endothelial cell line proliferation and migration (two functional bioassays), as well as Western blots, Northern blots, immunocytochemistry, and in situ hybridization. Data from these studies will provide the first framework for understanding the role of the renin angiotensin system's local control of and interactions between angiogenesis and blood flow to the uterine and placental vasculature, which are critical for normal fetal growth and development.
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