| The pregnancy-associated rise in estrogen is associated with a dramatic increase in utero-placental blood flow, and therefore laminar shear stress, a response required for normal fetal development. Estrogen is believed to exert its effect by binding to its specific estrogen receptors (ER) in target cells, resulting in increased expression and activity of endothelial nitric oxide synthase (eNOS). To examine the expression, cellular localization, and regulation of ERs in ovine UAs, vessels were fixed and mounted for mRNA and protein analysis. ER mRNA localization was determined by in situ hybridization. UA endothelial protein (UAendo) was isolated and the presence of ER protein was determined via Western blotting. Isolated UAECs were then cultured and treated with exogenous steroids, and analyzed using Western blotting, RT-PCR, and Immunocytochemistry (ICC). Under static and shear stress conditions, the effects of E2beta on stimulated (VEGF, bFGF, ATP) phosphorylation of peNOS S 1177, peNOS T495, pERK1/2, and pAkt were also studied. UAECs were grown on specialized cartridges and subjected to physiologic shear stress (15 dynes/cm2) in the presence (10nM) or absence of E2beta. Results. We observed by IHC and ISH that ERalpha and ERbeta protein and mRNA were present in ovine UAendo. Immunoblotting and RT-PCR showed expression of ERalpha and ERbeta protein and mRNA in UAECs. In contrast to static UAEC cultures, E2beta dramatically increased the expression of eNOS protein in the presence of shear stress. Furthermore, UAECs subjected to shear forces demonstrated a greater degree of phosphorylation of eNOS, Akt, and ERK when they are treated with E2beta than cells only subjected to shear stress Conclusion. Taken together, we show that ER protein and mRNA are expressed in UA endothelium and are regulated both by exogenous treatment with ovarian steroids, indicating UA endothelium is a target for estrogen action in vivo, and by novel signaling mechanisms probably initiated through serum and shear stress responsive elements. This is consistent with our theory of "programmed adaptive responses" to estrogen under changing laminar shear forces via exposure to pulsatile flows, which closely mimics the in vivo physiological changes in UBF. |
