Summary/Abstract The molecular mechanism of pathogenesis of preeclampsia (PE) is largely unknown, and effective prevention and treatment strategies remain elusive. PE is a pregnancy-associated hypertensive condition and complicates approximately one in 20 pregnancies in the US and is a leading cause of pregnancy-related maternal mortality and neonatal morbidity/mortality worldwide. Endothelin-1 (ET-1) is a vasoconstrictive peptide of 21 residues, and single nucleotide polymorphisms (SNPs) in EDN1, coding for a precursor for ET-1, are associated with PE. Edn1H/+ mice in which Edn1 expression is elevated to 3X normal have normal blood pressure, despite elevated circulating ET-1. However, Edn1H/+ dams develop full spectrum of PE-like phenotypes in their late pregnancy. In addition, the embryos from Edn1H/+ dams, regardless of their Edn1 genotypes, lag in development during early implantation stage with disoriented ectoplacental cones. We reported that nicotinamide (amide form of vitamin B3, Nam), inhibitor of ET-1 downstream of ADP ribosylcyclase, ameliorates the PE-like phenotypes in two separate mouse models of PE, and our preliminary data show that Nam decreases urinary albumin excretion and increases the number of survival fetuses when Edn1H/+ dams were treated during the entire pregnancy. These observations have led us to hypothesize that PE in Edn1H/+ dams originates from abnormal placentation caused by a high maternal ET-1 expression at early implantation stage, and that Nam can correct this damage and protect dams from later PE development. Accordingly, Specific Aim 1 will test this hypothesis by dissociating early effects from later effects of Nam on PE of Edn1H/+dams by treatments with this vitamin starting at different gestational stages and for different durations. Pregnancy outcomes including blood pressure, urinary albumin and fetal number and weight will be determined at 18.5 day post coitus (dpc). In addition, the expression of components of ET-1 system and Nam’s effects on them at implantation stage will be examined. Specific Aim 2 will investigate the mechanism of effects of ET-1 and Nam on differentiation from human trophoblast stem cells into designated trophoblast cells by using 2- and 3- dimension culture system. Pharmacological dose of ET-1 alone, or ET-1 plus Nam will be added to the specific conditioned medium. Cells will be examined by their morphology, motility, and expression of markers of different types of trophoblasts. Specific Aim 3 will investigate the mechanism of effects of ET-1 and Nam on impaired uterine decidualization and angiogenesis. The markers of endometrial stomal differentiation, the structure of blood vessels and the expression of vascular endothelial growth factor (VEGF) in uteri at the implantation stage of pregnancy will be examined. Primary cultured endometrial stromal cells will be treated with pharmacological dose of ET-1 alone, or ET-1 plus Nam, and the markers of differentiation and the expressio...