Formation of S-nitrothiols via addition of nitric oxide (NO)-derived nitrosyl groups to cysteines regulates the function of a plethora of proteins. This post-translational protein modification has been named as S- nitrosylation (S-NO) whose biological significance has been proposed to be analogous to phosphorylation. It represents an emerging physiological signaling mechanism that NO directly affects proteins and their functions. In this revised R21 application we are aimed at identifying novel endothelial cellular and mitochondrial SNO-proteins and at determining the functional sequelae of SNO-cofilin-1 and mechanisms linked to ER and endogenous NO via eNOS by using primary uterine artery endothelial cells and human umbilical vein endothelial cells as the models.
Aim 1 : To analyze estrogen responsive endothelial cellular nitrosyl-proteome by CyDye Switch, 2D two-dimensional fluorescence difference gel electrophoresis (2D- DIGE) and matrix-assisted laser desorption/ionization-time of flight (MALDI/TOF) mass spectrometry.
Aim 2 : To determine the subcellular localization of SNO-proteins in response to E22 and to analyze endothelial mitochondrial nitrosyl-proteome by CyDye Switch, 2D-DIGE and MALDI-TOF.
Aim 3 : To determine if estrogen-induced S-NO of proteins such as cofilin-1 is mediated by specific ER and endogenous NO via eNOS mediated mechanism(s).
Aim 4 : To determine if SNO-cofilin-1 regulates endothelial cell actin reorganization and migration by estrogen. This research is built on extensive in vitro and in vivo studies showing a stimulatory effect of estrogens on endothelial eNOS expression and NO production;however, the proposed studies are the first to take the next step for determining the downstream cellular and physiological processes that increased NO production by estrogen stimulation directly affects proteins and their functions and thus are of critical biological significance in estrogen, NO and endothelial biology. These studies are important in perinatal medicine as endothelium/NO dependent vasodilatation is a key mechanism responsible for estrogen-induced and pregnancy-associated rises in uterine blood flow that directly correlates to fetal development/survival and perinatal/neonatal outcomes. This research also will advance our understanding of the protective effects of estrogens in the cardiovascular system.

Public Health Relevance

This R21 aims to analyze endothelial cellular and mitochondrial nitrosyl-proteomes in hopes of identifying novel estrogen responsive nitrosylated protein targets and to investigate the role of cofilin-1 nitrosylation in endothelial cell actin organization/cell migration. The research takes the first next step for determining the downstream events that increased nitric oxide production by estrogen stimulation directly affects proteins and their functions. Data gained will provide knowledge leaps in understanding estrogen-induced and pregnancy associated uterine vasodilation critical for fetal development/survival and perinatal/neonatal outcomes and also relevant to estrogen protection of the cardiovascular system.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Exploratory/Developmental Grants (R21)
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Study Section
Pregnancy and Neonatology Study Section (PN)
Program Officer
Thrasher, Terry N
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University of California Irvine
Obstetrics & Gynecology
Schools of Medicine
United States
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Lechuga, Thomas J; Bilg, Amanpreet K; Patel, Bansari A et al. (2018) Estradiol-17? stimulates H2 S biosynthesis by ER-dependent CBS and CSE transcription in uterine artery smooth muscle cells in vitro. J Cell Physiol :
Chen, Dong-Bao; Feng, Lin; Hodges, Jennifer K et al. (2017) Human trophoblast-derived hydrogen sulfide stimulates placental artery endothelial cell angiogenesis. Biol Reprod 97:478-489
Zhang, Hong-Hai; Chen, Jennifer C; Sheibani, Lili et al. (2017) Pregnancy Augments VEGF-Stimulated In Vitro Angiogenesis and Vasodilator (NO and H2S) Production in Human Uterine Artery Endothelial Cells. J Clin Endocrinol Metab 102:2382-2393
Sheibani, Lili; Lechuga, Thomas J; Zhang, Honghai et al. (2017) Augmented H2S production via cystathionine-beta-synthase upregulation plays a role in pregnancy-associated uterine vasodilation. Biol Reprod 96:664-672
Zhang, Hong-Hai; Lechuga, Thomas J; Chen, Yuezhou et al. (2016) Quantitative Proteomics Analysis of VEGF-Responsive Endothelial Protein S-Nitrosylation Using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and LC-MS/MS. Biol Reprod 94:114
Lechuga, Thomas J; Zhang, Hong-hai; Sheibani, Lili et al. (2015) Estrogen Replacement Therapy in Ovariectomized Nonpregnant Ewes Stimulates Uterine Artery Hydrogen Sulfide Biosynthesis by Selectively Up-Regulating Cystathionine ?-Synthase Expression. Endocrinology 156:2288-98
Zhang, Hong-Hai; Wang, Wen; Feng, Lin et al. (2015) S-nitrosylation of Cofilin-1 Serves as a Novel Pathway for VEGF-Stimulated Endothelial Cell Migration. J Cell Physiol 230:406-17
Chen, Dong-Bao; Zheng, Jing (2014) Regulation of placental angiogenesis. Microcirculation 21:15-25
Satohisa, Seiro; Zhang, Hong-hai; Feng, Lin et al. (2014) Endogenous NO upon estradiol-17? stimulation and NO donor differentially regulate mitochondrial S-nitrosylation in endothelial cells. Endocrinology 155:3005-16
Chen, Dong-bao; Wang, Wen (2013) Human placental microRNAs and preeclampsia. Biol Reprod 88:130

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