IMPLANTATION AND PREGNANCY - A
Monday, August 2, 2004
10:30 AM–12:30 PM
(245) ESTROGEN RECEPTORS AND VEGF MODULATION OF ANGIOGENIC AND NITRIC OXIDE RESPONSES IN OVINE UTERINE ARTERY ENDOTHELIAL CELLS.
Magness, Ronald1, 2, 3, Byers, Michael1, King, Adam1, Austin, Jason1, Yi, FuXian1, Bird, Ian1, Zheng, Jing1, 1 University Of Wisconsin, Madison, WI2 University of Wiscosnin, Madison, WI3 University of Wisconsin, Madison, WI
ABSTRACT- Uterine blood flow (UBF) is substantially elevated during high estrogen states such as the follicular phase and pregnancy. Follicular and Pregnancy rises in UBF are inhibited, in vivo, by estrogen receptor (ER) antagonist ICI 182,780 and NOS inhibitor L-NAME demonstrating endogenous E2B-induced increases in UBF via a NO mediated mechanism. Although both NO-mediated vasodilatation and VEGF-associated angiogenesis appear to control UBF, the mechanistic basis and interactions for this are still uncertain. We used in situ hybridization (ISH) ([35S]-labeled riboprobes) and immunohistochemistry (IHC) for mRNA and protein localization. The presence of both ERa and ERB mRNA and protein was identified in uterine artery endothelium ex vivo. In passage 4-5 UAECs both ERa and ERB mRNA and protein were also observed, suggesting that UAECs may be a good model to evaluate direct actions of E2B on the uterine vascular bed. We observed that E2B dose dependently elevated VEGF secretion by cultures of UAECs with maximum responses of 3-4 fold at 10nM (P<0.001). This E2B-mediated VEGF secretion was associated with elevations in UAEC angiogenesis using a capillary-like tube formation assay. E2B-induced rises in VEGF and angiogenesis were blocked by ICI 182,780 demonstrating ER receptor involvement. Since the E2B-induced rises in VEGF may also increase vasodilatation, we tested the hypothesis that NO production by UAECs in response to E2B would be similar to VEGF responses. Employing a newly developed real-time intracellular NO assay using DAF-FM DA in a fluorescent plate reader, we observed that in UAECs both E2B (10nM) and VEGF (10ng/ml) increased NO production 2.0 and 1.5 fold, respectively above control; the combination was neither synergistic nor additive. Since the slope of the time course rise in NO with E2B was much somewhat steeper than the observed rise with VEGF, it is unlikely that the E2B-induced rise in NO is via stimulation of VEGF. In contrast both sFlt-1 and Flt-1 blocking antibody inhibited the E2B-mediated rise in angiogenesis. Thus based on this in vitro UAEC model E2B-associated rises in VEGF appear to help modulate angiogenesis, but not NO-mediated vasodilatation. NIH HL49210, HD33255, HL57653, HD38843, HL64703, & HL64601.
KEY WORDS: VEGF, Angiogenesis, Estrogen, Nitric Oxide