Normal pregnancy is associated with dramatic increases in uteroplacental and fetoplacental blood flows directly correlating with fetal growth, survival and neonatal birth weights. These increases in placental blood flows result from both neovascularization and vasodilatation. During the third trimers of ovine and human pregnancy, at a time when fetal growth and uteroplacental and feto-placental blood flows increase exponentially, the maternal and fetal compartments produce angiogenic factors such as bFGF and VEGF, as well as Nitric Oxide (NO). Elevations in blood flow also substantially increase the laminar shear stress on the endothelial cells, thus the increase in angiogenesis and NO production are associated with a time of pregnancy when neovascularization and vasodilatation are observed in the uterine angiogenesis and NO production are associated with a time of pregnancy when neovascularization and vasodilatation are observed in the uterine and fetoplacental vasculature and shear stress is increasing. The hypothesis of this proposal is that the placental and uterine-derived angiogenic factors, bFGF and VEGF serve to augment or modulate shear stress induced increases in NO production and eNOS expression by placental factors, bFGF and VEGF serve to augment or modulate shear stress induced increases in NO production and eNOS expression by placental and uterine artery endothelium so as to control blood flow at the utero-placental interface. In two Specific Aims we will use endothelial cells grown to confluence on artificial capillary beds in CellMax cartridges, a novel model we have developed recently in our laboratory.
Specific Aim 1 : Angiogenic growth factor (bFGF or VEGF) interaction with shear stress on NO production, eNOS expression (protein and mRNA) and the rate of de novo mRNA transcription in ovine fetoplacental artery endothelial cells (OFPAEC) and uterine artery endothelial cells (UAEC) from pate pregnant and non-pregnant sheep. We will also evaluate the effects of laminar shear stress on bFGF and VEGF production/expression and the expression of their receptors (FGFR-1/Flg-1, Flk-1/KDR). Effects of Actinomycin D or Cyclohexamide on the synergistic actions of angiogenic growth factors (bFGF or VEGF) and laminar shear stress in OFPAEC and UAEC NO production, eNOS protein and mRNA expression.
Specific Aim 2 : Effects of inhibition of specific signaling pathways on angiogenic growth factor (bFGF or VEGF)-shear stress induced elevations in NO production and eNOS expression; (2a) Mitogen Activated Protein Kinase (MAPK) signaling pathways (ERK +, p38MAPK) and SAPK/JNK pathway); (2b) Calcium signaling pathway; (2b) Calcium signaling pathway; (2c) Protein kinase C signaling pathway (a possible MEK dependent pathway); (2d)m PI3-Kinase/AKT signaling pathway (a MEK independent pathway). Data derived from these studies will provide the first framework for understanding the role of the local control and interactions between shear stress, angiogenesis, and NO to control blood flow to the placental and uterine vasculature, which are critical for normal fetal growth and development. Furthermore, understanding differences and similarities between the fetal and maternal endothelial cell responses to angiogenic factors also will allow us to understand how both sides of the placental communicate during this important period of fetal growth. The importance of these proposed studies become very evident since the normally dramatic increases in fetoplacental and uteroplacental perfusion are directly linked to fetal growth and that these mechanisms are dysfunctional in pathologic pregnancies such as preeclampsia and IUGR. These mechanisms thus relevant to perinatal medicine since modulation of fetal growth impacts fetal survivability.
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