PROJECT SUMMARY Once conceived, a woman’s cardiovascular system undergoes dramatic structural and functional changes to accommodate the increasing demands of the fast growing fetus, resulting in profound uterine artery dilation exemplified by dramatic rise in uterine blood flow (UBF). UBF is a rate-limiting factor for pregnancy health because an insufficient rise in UBF during pregnancy is causative for intrauterine growth restriction and preeclampsia (PE) characterized by systemic endothelial damage and vascular dysfunction. Since 1900’s, numerous studies have concluded that local endothelial nitric oxide (NO)-mediated vasodilation is the major mechanism controlling rise in UBF. However, blockade of local NO production only partially inhibits baseline pregnancy-associated rise in UBF, suggesting that other mediator(s) are involved. Endogenous hydrogen sulfide (H2S), mainly synthesized from L-cysteine by two key enzymes: cystathionine -synthase (CBS) and cystathionine -lyase (CSE), is an extremely potent proangiogenic vasodilator. We initially posited that local CBS/H2S production can fill the mechanism behind NO to mediate UA vasodilation during pregnancy. Indeed, we reported that pregnancy dramatically augments UA H2S biosynthesis by selectively upregulating EC and SM CBS but not CSE expression in animals (rats and ewes) and women in vivo and that H2S stimulates pregnancy-dependent dilation of pressurized UA ex vivo. In animal models of PE and women with PE, we found that pregnancy-augmented myometrial UA CBS/H2S is significantly downregulated. However, research on H2S in uterine hemodynamics is still in its infancy; many important key questions need to be answered before a physiological and a pathophysiological role of CBS/H2S signaling in normal pregnancy and PE can be determined. In this new RO1 we propose to test a novel hypothesis that enhanced UA EC and SM CBS/H2S production mediates pregnancy-associated UA dilation by interacting with vascular endothelial growth factor and EC eNOS-NO and downregulated UA CBS/H2S signaling contributes to the vascular dysfunction in PE. We will test this hypothesis by a multidisciplinary translational approach with biochemical, cellular, molecular, physiological, and pharmacological methods coupled with rat models in vivo, freshly isolated human and rat UA rings ex vivo, novel human UA EC (hUAEC) and smooth muscle cell (hUASMC) models in vitro, and myometrial UAs from normotensive vs. PE pregnant women. We have an outstanding team with a track record of long-term productive collaborative research in the field and unique tools needed to complete this exciting and important project. We believe that the novel studies outlined in this RO1 will provide new data to fill a knowledge gap on the physiological and pathophysiological role for H2S in in uterine hemodynamic regulation and this knowledge will provide a compelling rationale for clinical trials to explore the therapeutic potential of H2S in women in hig...