Interactions between estrogen and NO to regulate UBF at the maternal-fetal placental interface are critically important to fetal growth. Pregnant (P) and Follicular (Fol) Nonpregnant (NP) Uterine Artery Endothelial Cells (UAEC) undergo adaptation compared to Luteal (Lut) NP-UAEC; i.e. responses seen in freshly isolated UA Endothelium maintaining upon numerous passages. These include ERK 1/2 -MAPK coupling to eNOS activation for substantial ATP-stimulated NO production (P > Fol-NP> Lut-NP) and the role estrogen may have to alter Ca2+ mediated NO. Shear stress and/or estrogen are both very potent stimulators of NO synthesis and/or eNOS expression and together they elicit synergistic activation of the ERK 1/2 MAPK signaling pathway in P-UAEC. Elevations in blood flow also substantially increase the laminar shear stress. Other signaling pathways (e.g. PI3 Kinase) may play an additional convergent role in the in vivo situation. Overall hypothesis: Physiologic states of High DBF/shear stress and elevated estrogen (e.g. P and Fol) causes """"""""Programmed Adaptation"""""""" of the UAEC to modulate important endothelial functions associated with NO production. Specific emphasis is given to mechanisms that increase eNOS activation, eNOS expression, UAEC signaling kinases (e.g. ERK 1/2 and/or. PIS Kinase), and cell-cell communication (measured as synchronized Ca2+ bursts).
Aim I - Physiologic Models: Using In V/Vo-derived freshly isolate UA Endothelium (Passage 0) from Pregnant, Cycling (Fol and Lut) and Ovariectomized (Vehicle vs Estrogen-treated) sheep, studies will be performed evaluating cellular regulation (nongenomic and genomic) by which Estrogen alters basal, ATP and lonomycin eNOS activation by directly imaging intracellular NO production simultaneously with [Ca2+]i/Ca2+ bursts and evaluating the role of ERs and converging signaling intermediate (ERK1/2 MAPK and/or PIS Kinase) pathways.
Aim II - Shear Stress Models: Effects of prolonged Laminar Shear Stress [0 (static), 3 & 15 dynes/cm2] with and without E2(3 on """"""""Programmed UAEC Adaptation"""""""" of long term cultured (Passage 4-5) Lut NP-UAEC, Fol NP- UAEC, and P-UAEC. The specific markers of adaptation that are """"""""programmed"""""""" from the time isolation we focus on include: a) eNOS expression and ATP/lonomycin-stimulated eNOS activation and [Ca2+]i/Ca2+ bursts and b) ATP/lonomycin-stimulated ERK1/2 and AKT phosphorylation; and inhibition of ERK-MAPK and/or PIS Kinase mediated stimulated NO production and [Ca2+]i/Ca2+ bursts. We hypothesize that inclusion of both shear stress and estrogen will be necessary to restore the specific markers and response magnitude """"""""programmed"""""""" into UAECs by high flow and estrogen during Pregnancy and in the Follicular phase defined in Aim I. Data thus derived will provide the first mechanistic framework for understanding interactions between shear stress and estrogen to regulate UAEC NO and converging signaling mechanisms this. Our recent preliminary data demonstrate that uterine artery shear stress is indeed reduced in pregnancies complicated by preeclampsia. Therefore these mechanisms are likely also to be dysfunctional during preeclampsia with or without IUGR. ? ? ?
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