With potent vasodilatory and pro-angiogenic properties, hydrogen sulfide (H₂S) is now accepted as the third gasotransmitter after nitric oxide (NO) and carbon monoxide. Endogenous H₂S is mainly synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). Akin to previous studies showing hormonal regulation of NO biosynthesis, we first reported that uterine and systemic artery H₂S biosynthesis is regulated by exogenous estrogens in an ovariectomized sheep model of estrogen replacement therapy, specifically stimulating CBS, but not CSE, expression, in uterine (UA) and mesenteric (MA), but not carotid (CA), arteries in ovariectomized nonpregnant sheep. We have found significantly elevated H₂S biosynthesis due to CBS upregulation under estrogen-dominant physiological states, the proliferative phase of menstrual cycle and pregnancy in primary human UAs. Our studies have pioneered the role of H₂S biology in uterine hemodynamics regulation although there is still much that needs to be learned before a thorough elucidation of a role that H₂S plays in normal physiology of uterine hemodynamics and its dysregulation under pregnancy complications can be determined. In this chapter we describe a series of methods that we have optimized for analyzing vascular H₂S biosynthesis, including (1) real-time quantitative PCR (qPCR) for assessing tissue and cellular levels of CBS and CSE mRNAs, (2) immunoblotting for assessing CBS and CSE proteins, (3) semiquantitative immunofluorescence microscopy to specifically localize CBS and CSE proteins on vascular wall and to quantify their cellular expression levels, and (4) methylene blue assay for assessing H₂S production in the presence of selective CBS and CSE inhibitors.

Endogenous hydrogen sulfide (H2S) is a potent vasodilator and proangiogenic second messenger synthesized from L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). Estrogens are potent vasodilators that stimulate H2S biosynthesis in uterine arteries (UA) in vivo; however, the underlying mechanisms are unknown. We hypothesized that estrogens stimulate H2S biosynthesis in UA endothelial cells (UAEC) via specific estrogen receptor (ER)-dependent mechanisms. In cultured primary UAEC, treatment with estradiol-17β (E2β) stimulated CBS and CTH mRNAs and proteins in a time- and concentration-dependent fashion. As little as 0.1 nM E2β was effective in increasing CBS and CTH expressions and these stimulatory effects maximized with 10-100 nM E2β at 48-72 h. E2β also activated CBS and CTH promoters in UAEC, leading to CBS and CTH expression. Treatment with E2β stimulated H2S production, which was blocked by specific inhibitors of either CBS or CTH and their combination and the ER antagonist ICI 182780. Treatment with either specific agonist of ERα or ERβ stimulated both CBS and CTH mRNA and protein expressions and H2S production to levels similar to that of E2β. Specific antagonist of either ERα or ERβ blocked E2β-stimulated CBS and CTH mRNA and protein expressions and H2S production. Combinations of either ERα or ERβ agonists or their antagonists had no additive effects. Thus, E2β stimulates H2S production by upregulating CBS and CTH mRNA and protein expressions through specific ERα or ERβ-dependent CBS and CTH transcription in UAEC in vitro.

Context

Augmented uterine artery (UA) production of vasodilators, including nitric oxide (NO) and hydrogen sulfide (H2S), has been implicated in pregnancy-associated and agonist-stimulated rise in uterine blood flow that is rate-limiting to pregnancy health.

Objective

Developing a human UA endothelial cell (hUAEC) culture model from main UAs of nonpregnant (NP) and pregnant (P) women for testing a hypothesis that pregnancy augments endothelial NO and H2S production and endothelial reactivity to vascular endothelial growth factor (VEGF).

Design

Main UAs from NP and P women were used for developing hUAEC culture models. Comparisons were made between NP- and P-hUAECs in in vitro angiogenesis, activation of cell signaling, expression of endothelial NO synthase (eNOS) and H2S-producing enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase, and NO/H2S production upon VEGF stimulation.

Results

NP- and P-hUAECs displayed a typical cobblestone-like shape in culture and acetylated low-density lipoprotein uptake, stained positively for endothelial and negatively for smooth muscle markers, maintained key signaling proteins during passage, and had statistically significant greater eNOS and CBS proteins in P- vs NP-hUAECs. Treatment with VEGF stimulated in vitro angiogenesis and eNOS protein and NO production only in P-hUEACs and more robust cell signaling in P- vs NP-hUAECs. VEGF stimulated CBS protein expression, accounting for VEGF-stimulated H2S production in hUAECs.

Conclusion

Comparisons between NP- and P-hUAECs reveal that pregnancy augments VEGF-stimulated in vitro angiogenesis and NO/H2S production in hUAECs, showing that the newly established hUAEC model provides a critical in vitro tool for understanding human uterine hemodynamics.

Rapid nitric oxide (NO) production via endothelial NO synthase (eNOS) activation represents a major signaling pathway for the cardiovascular protective effects of estrogens; however, the pathways after NO biosynthesis that estrogens use to function remain largely unknown. Covalent adduction of a NO moiety to cysteines, termed S-nitrosylation (SNO), has emerged as a key route for NO to directly regulate protein function. Cofilin-1 (CFL1) is a small actin-binding protein essential for actin dynamics and cytoskeleton remodeling. Despite being identified as a major SNO protein in endothelial cells, whether SNO regulates CFL-1 function is unknown. We hypothesized that estradiol-17β (E2β) stimulates SNO of CFL1 via eNOS-derived NO and that E2β-induced SNO-CFL1 mediates cytoskeleton remodeling in endothelial cells. Point mutation studies determined Cys80 as the primary SNO site among the 4 cysteines (Cys39/80/139/147) in CFL1. Substitutions of Cys80 with Ala or Ser were used to prepare the SNO-mimetic/deficient (C80A/S) CFL1 mutants. Recombinant wild-type (wt) and mutant CFL1 proteins were prepared; their actin-severing activity was determined by real-time fluorescence imaging analysis. The activity of C80A CFL1 was enhanced to that of the constitutively active S3/A CFL1, whereas the other mutants had no effects. C80A/S mutations lowered Ser3 phosphorylation. Treatment with E2β increased filamentous (F)-actin and filopodium formation in endothelial cells, which were significantly reduced in cells overexpressing wt-CFL. Overexpression of C80A, but not C80S, CFL1 decreased basal F-actin and further suppressed E2β-induced F-actin and filopodium formation compared with wt-CFL1 overexpression. Thus, SNO(Cys80) of cofilin-1 via eNOS-derived NO provides a novel pathway for mediating estrogen-induced endothelial cell cytoskeleton remodeling.

Adduction of a nitric oxide moiety (NO•) to cysteine(s), termed S-nitrosylation (SNO), is a novel mechanism for NO to regulate protein function directly. However, the endothelial SNO-protein network that is affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture for comparing vascular endothelial growth factor (VEGFA)- and NO donor-responsive endothelial nitroso-proteomes. Primary placental endothelial cells were labeled with "light" (L-(12)C6 (14)N4-Arg and L-(12)C6 (14)N2-Lys) or "heavy" (L-(13)C6 (15)N4-Arg and L-(13)C6 (15)N2-Lys) amino acids. The light cells were treated with an NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the heavy cells received vehicle as control. Equal amounts of cellular proteins from the light (GSNO or VEGFA treated) and heavy cells were mixed for labeling SNO-proteins by the biotin switch technique and then trypsin digested. Biotinylated SNO-peptides were purified for identifying SNO-proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ratios of light to heavy SNO-peptides were calculated for determining the changes of the VEGFA- and GSNO-responsive endothelial nitroso-proteomes. A total of 387 light/heavy pairs of SNO-peptides were identified, corresponding to 213 SNO-proteins that include 125 common and 27 VEGFA- and 61 GSNO-responsive SNO-proteins. The specific SNO-cysteine(s) in each SNO-protein were simultaneously identified. Pathway analysis revealed that SNO-proteins are involved in various endothelial functions, including proliferation, motility, metabolism, and protein synthesis. We collectively conclude that endogenous NO on VEGFA stimulation and exogenous NO from GSNO affect common and different SNO-protein networks, implicating SNO as a critical mechanism for VEGFA stimulation of angiogenesis.

Uterine vasodilation dramatically increases during the follicular phase of the estrous cycle and pregnancy, which are estrogen-dominant physiological states. Uterine vasodilation is believed to be mainly controlled by local uterine artery (UA) production of vasodilators and angiogenic factors. The extremely potent vasodilator and proangiogenic hydrogen sulfide (H2S) is synthesized via metabolizing L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). This study was designed to determine if UA H2S production increases with augmented expression and/or activity of CBS and/or CTH during the ovarian cycle and pregnancy in sheep. Uterine arteries from intact nonpregnant (NP) luteal and follicular phase and late (130-135 days, term ≈ 145 days) pregnant (P) ewes were collected; endothelium-enriched proteins (UAendo) and endothelium-denuded smooth muscle (UAvsm) were mechanically prepared for accessing CBS and CTH proteins by immunoblotting; their cellular localization was determined by semi-quantitative immunofluorescence microscopy. H2S production was measured by the methylene blue assay. Immunoblotting revealed that CBS but not CTH protein was greater in P > > > NP follicular > luteal UAendo and UAvsm (P < 0.001). H2S production was greater in P > > > NP UAendo and UAvsm (P < 0.01). Pregnancy-augmented UAendo and UAvsm H2S production was inhibited by the specific CBS but not CTH inhibitor. CBS and CTH proteins were localized to both endothelium and smooth muscle; however, only CBS protein was significantly greater in P vs NP UA endothelium and smooth muscle. Thus, ovine UA H2S production is significantly augmented via selectively upregulating endothelium and smooth muscle CBS during the follicular phase and pregnancy in vivo.

Endogenous hydrogen sulfide (H₂ S), synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), is a potent vasodilator that can be stimulated by estradiol-17β (E ₂ β) in uterine artery (UA) smooth muscle (UASMC) in vivo; however, the underlying mechanisms are unknown. This study tested a hypothesis that E ₂ β stimulates H ₂ S biosynthesis by upregulating CBS expression via specific estrogen receptor (ER). Treatment with E ₂ β stimulated time- and concentration- dependent CBS and CSE messenger RNA (mRNA) and protein expressions, and H ₂ S production in cultured primary UASMC isolated from late pregnant ewes, which were blocked by ICI 182,780. Treatment with specific ERα or ERβ agonist mimicked these E ₂ β-stimulated responses, which were blocked by specific ERα or ERβ antagonist. Moreover, E ₂ β activated both CBS and CSE promoters and ICI 182,780 blocked the E ₂ β-stimulated responses. Thus, E ₂ β stimulates H ₂ S production by upregulating CBS and CSE expression via specific ER-dependent transcription in UASMC in vitro.

Endogenous hydrogen sulfide (H2S), mainly synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH), has been implicated in regulating placental angiogenesis; however, the underlying mechanisms are unknown. This study was to test a hypothesis that trophoblasts synthesize H2S to promote placental angiogenesis. Human choriocarcinoma-derived BeWo cells expressed both CBS and CTH proteins, while the first trimester villous trophoblast-originated HTR-8/SVneo cells expressed CTH protein only. The H2S producing ability of BeWo cells was significantly inhibited by either inhibitors of CBS (carboxymethyl hydroxylamine hemihydrochloride, CHH) or CTH (β-cyano-L-alanine, BCA) and that in HTR-8/SVneo cells was inhibited by CHH only. H2S donors stimulated cell proliferation, migration, and tube formation in ovine placental artery endothelial cells (oFPAECs) as effectively as vascular endothelial growth factor. Co-culture with BeWo and HTR-8/SVneo cells stimulated oFPAEC migration, which was inhibited by CHH or BCA in BeWo but CHH only in HTR-8/SVneo cells. Primary human villous trophoblasts (HVT) were more potent than trophoblast cell lines in stimulating oFPAEC migration that was inhibited by CHH and CHH/BCA combination in accordance with its H2S synthesizing activity linked to CBS and CTH expression patterns. H2S donors activated endothelial nitric oxide synthase (NOS3), v-AKT murine thymoma viral oncogene homolog 1 (AKT1), and extracellular signal-activated kinase 1/2 (mitogen-activated protein kinase 3/1, MAPK3/1) in oFPAECs. H2S donor-induced NOS3 activation was blocked by AKT1 but not MAPK3/1 inhibition. In keeping with our previous studies showing a crucial role of AKT1, MAPK3/1, and NOS3/NO in placental angiogenesis, these data show that trophoblast-derived endogenous H2S stimulates placental angiogenesis, involving activation of AKT1, NOS3/NO, and MAPK3/1.

Elevated endogenous estrogens stimulate human uterine artery endothelial cell (hUAEC) hydrogen sulfide (H2S) production by selectively upregulating the expression of H2S synthesizing enzyme cystathionine β-synthase (CBS), but the underlying mechanisms are underdetermined. We hypothesized that CBS transcription mediates estrogen-stimulated pregnancy-dependent hUAEC H2S production. Estradiol-17β (E2β) stimulated CBS but not cystathionine γ-lyase (CSE) expression in pregnant human uterine artery ex vivo, which was attenuated by the estrogen receptor (ER) antagonist ICI 182,780. E2β stimulated CBS mRNA/protein and H2S production in primary hUAEC from nonpregnant and pregnant women, but with greater responses in pregnant state; all were blocked by ICI 182,780. Human CBS promoter contains multiple estrogen-responsive elements (EREs), including one ERE preferentially binding ERα (αERE) and three EREs preferentially binding ERβ (βERE), and one full ERE (α/βERE) and one half ERE (½α/βERE) binding both ERα and ERβ. Luciferase assays using reporter genes driven by human CBS promoter with a series of 5'-deletions identified the α/βEREs binding both ERα and ERβ (α/βERE and ½α/βERE) to be important for baseline and E2β-stimulated CBS promoter activation. E2β stimulated ERα/ERβ heterodimerization by recruiting ERα to α/βEREs and βERE, and ERβ to βERE, α/βEREs, and αERE. ERα or ERβ agonist alone trans-activated CBS promoter, stimulated CBS mRNA/protein and H2S production to levels comparable to that of E2β-stimulated, while ERα or ERβ antagonist alone abrogated E2β-stimulated responses. E2β did not change human CSE promoter activity and CSE mRNA/protein in hUAEC. Altogether, estrogen-stimulated pregnancy-dependent hUAEC H2S production occurs by selectively upregulating CBS expression via ERα/ERβ-directed gene transcription.

The pregnancy-augmented uterine vasodilation is linked to increased AT₂R (angiotensin type-2 receptor) that mediates the vasodilatory effects of angiotensin II. However, the mechanisms controlling AT₂R expression during pregnancy remain unclear. Estrogens are known to play a role in vascular adaptations during pregnancy. We hypothesized that estrogen stimulates uterine artery AT₂R expression via ER (estrogen receptor)-β-dependent transcription in a pregnancy-specific endothelium-dependent manner. Plasma estradiol levels increased and peaked in late pregnancy and returned to prepregnant levels post-partum, correlating with uterine artery AT₂R and ERβ upregulation. Estradiol stimulated AT₂R mRNA expression in endothelium-intact but not endothelium-denuded late pregnant and nonpregnant rat uterine artery ex vivo. Consistently, estradiol stimulated AT₂R mRNA expression in late pregnant but not nonpregnant primary human uterine artery endothelial cells in vitro, which was abolished by ER antagonist ICI 182,780. Higher ERα protein bound to ER-responsive elements in AT₂R promoter in the nonpregnant arteries whereas higher ERβ bound in the pregnant state. ERα protein levels were similar but higher ERβ protein levels were expressed in pregnant versus nonpregnant human uterine artery endothelial cells. Estradiol stimulation recruited ERα to the AT₂R promoter in the nonpregnant state and ERβ to the AT₂R promoter in pregnancy; however, only ERβ recruitment mediated transactivation of the AT₂R reporter gene in pregnant human uterine artery endothelial cells. Estradiol-induced AT₂R expression was abolished by the specific ERβ (not ERα) antagonist 4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP) and mimicked by the specific ERβ (not ERα) agonist 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN) in pregnant human uterine artery endothelial cells in vitro. This study demonstrates a novel role of pregnancy-augmented ERβ in AT₂R upregulation in the uterine artery and provides new insights into the mechanisms underlying uterine vascular adaptation to pregnancy.