Gladys E. Lopez

Endothelial vasodilator production by ovine uterine and systemic arteries: ovarian steroid and pregnancy control of ERα and ERβ levels

Pregnancy and the follicular phase are physiological states of elevated oestrogen levels and rises in uterine blood flow (UBF). The dramatic increase in utero‐placental blood flow during gestation is required for normal fetal growth and development. Oestrogen exerts its vasodilatory effect by binding to its specific oestrogen receptors (ER) in target cells, resulting in increased expression and activity of endothelial nitric oxide synthase (eNOS) to relax vascular smooth muscle (VSM). However, the regulation of endothelial versus VSM ERα and ERβ expression in uterine arteries (UAs) during the ovarian cycle, pregnancy and with exogenous hormone replacement therapy (HRT) are currently unknown. ER mRNA and protein localization was determined by in situ hybridization (ISH) using 35S‐labelled riboprobes and immunohistochemistry (IHC), respectively. UA endothelial (UAendo), UA VSM, omental artery endothelium (OA endo), and OA VSM proteins were isolated and ERα and ERβ protein expression was determined by Western analysis. We observed by ISH and IHC that ERα and ERβ mRNA and protein were localized in both UAendo and UA VSM. Immunoblot data demonstrated ovarian hormone specific regulation of ERα and ERβ protein in UAendo and UA VSM. Compared to luteal phase sheep, both ERα and ERβ levels in UAendo were elevated in follicular phase sheep. Whereas ERβ was elevated by pregnancy in UAendo and UA VSM, ERα was not appreciably altered. eNOS was increased in UAendo from follicular and pregnant sheep. Ovariectomized ewes (OVEX) had substantially reduced UAendo ERβ, but not UAendo ERα or OAendo ERα and ERβ. In contrast, OVEX increased UA VSM ERα and ERβ and decreased OA VSM ERα and ERβ. Treatment with oestradiol‐17β (E2β), but not progesterone or their combination, increased UAendo ERα levels. The reduced ERβ in UAendo from OVEX ewes was reversed by E2β and progesterone treatment. While ERα and eNOS were not elevated in any other reproductive or non‐reproductive endothelia tested, ERβ was augmented by pregnancy in uterine, mammary, placenta, and coronary artery endothelia. ERα and ERβ mRNA and protein are expressed in UA endothelium with expression levels depending on the endocrine status of the animal, indicating UA endothelium is a target for oestrogen action in vivo, and that the two receptors appear to be differentially regulated in a spatial and temporal fashion with regard to the reproductive status or HRT.

Endothelial caveolar hub regulation of adenosine triphosphate-induced endothelial nitric oxide synthase subcellular partitioning and domain-specific phosphorylation.

ATP leads to endothelial NO synthase (eNOS)/NO-mediated vasodilation, a process hypothesized to depend on the endothelial caveolar eNOS partitioning and subcellular domain-specific multisite phosphorylation state. We demonstrate herein that, in both the absence and presence of ATP, the uterine artery endothelial caveolae contain specific protein machinery related to subcellular partitioning and act as specific focal “hubs” for NO- and ATP-related proteins. ATP-induced eNOS regulation showed a complex set of multisite posttranslational phosphorylation events that were closely associated with the enzymes partitioning between caveolar and noncaveolar endothelial subcellular domains. The comprehensive model that we present demonstrates that ATP repartitioned eNOS between the caveolar and noncaveolar subcellular domains; specifically, the stimulatory PSer635eNOS was substantially higher in the caveolar pool with subcellular domain-independent increased levels on ATP treatment. The stimulatory PSer1179eNOS was not altered by ATP treatment. However, the inhibitory PThr495eNOS was regulated predominantly in the caveolar domain with decreased levels on ATP action. In contrast, the agonist-specific PSer114eNOS was localized in the noncaveolar pool with increased levels on ATP stimulation. Thus, the endothelial caveolar membrane system plays a pivotal role(s) in ATP-associated subcellular partitioning and possesses the relevant protein machinery for ATP-induced NO regulation. Furthermore, these subcellular domain-specific phosphorylation/dephosphorylation events provide evidence relating to eNOS spatio-temporal dynamics.

Local Effects of Pregnancy on Connexin Proteins That Mediate Ca2+-Associated Uterine Endothelial NO Synthesis

Uterine artery adaptations during gestation facilitate increases in uterine blood flow and fetal growth. Hypothesis: local expression and distribution of uterine artery connexins play roles in mediating in vivo gestational eNOS activation and NO production. We established an ovine model restricting pregnancy to a single uterine horn and measured uterine blood flow, uterine artery shear stress, connexins 37/43, and P635eNOS protein levels in uterine artery and systemic artery (omental and renal) endothelium and connexins in vascular smooth muscle. Uterine blood flow and shear stress were locally (unilaterally) and substantially elevated by gestation. During pregnancy, uterine artery endothelial gap junction proteins connexins 37/43 were locally regulated in the gravid horn and elevated 10.3- and 25.6-fold; uterine artery endothelial P635eNOS and total eNOS were elevated 3.3- and 2.9-fold; whereas uterine artery vascular smooth muscle connexins 37/43 were locally elevated 12.5- and 5.9-fold, respectively. Less pronounced changes were observed in systemic vasculature except for significant pregnancy-associated increases in omental artery vascular smooth muscle connexin 43 and omental artery endothelial P635eNOS and total eNOS. Gap junction blockade using connexin 43, but not connexin 37–specific Gap peptides, abrogated uterine artery endothelial ATP-induced Ca2+-mediated NO production. Thus, uterine artery endothelial connexin 43, but not connexin 37, regulates Ca2+-mediated NO production required for the vasodilation to accommodate increases in uterine blood flow and shear stress during healthy pregnancies.

Computational flow cytometry analysis reveals a unique immune signature of the human maternal‐fetal interface

Decidual immune dysregulation is thought to underlie major pregnancy disorders; however, incomplete understanding of the decidual immune interface has hampered the mechanistic investigation.

Convergent ERK1/2, p38 and JNK mitogen activated protein kinases (MAPKs) signalling mediate catecholoestradiol‐induced proliferation of ovine uterine artery endothelial cells

The catechol metabolites of 17β‐oestradiol (E2β), 2‐hydroxyoestradiol (2‐OHE2) and 4‐hydroxyoestradiol (4‐OHE2), stimulate proliferation of pregnancy‐derived ovine uterine artery endothelial cells (P‐UAECs) through β‐adrenoceptors (β‐ARs) and independently of the classic oestrogen receptors (ERs). Herein we show that activation of ERK1/2, p38 and JNK mitogen activated protein kinases (MAPKs) is necessary for 2‐OHE2‐ and 4‐OHE2‐induced P‐UAEC proliferation, as well as proliferation induced by the parent hormone E2β and other β‐AR signalling hormones (i.e. catecholamines). Conversely, although 2‐OHE2 and 4‐OHE2 rapidly activate phosphatidylinositol 3‐kinase (PI3K), its activation is not involved in catecholoestradiol‐induced P‐UAEC proliferation. We also show for the first time the signalling mechanisms involved in catecholoestradiol‐induced P‐UAEC proliferation; which converge at the level of MAPKs with the signalling mechanisms mediating E2β‐ and catecholamine‐induced proliferation. The present study advances our understanding of the complex signalling mechanisms involved in regulating uterine endothelial cell proliferation during pregnancy.

Decidual-Placental Immune Landscape During Syngeneic Murine Pregnancy

Adaptive immune system, principally governed by the T cells—dendritic cells (DCs) nexus, is an essential mediator of gestational fetal tolerance and protection against infection. However, the exact composition and dynamics of DCs and T cell subsets in gestational tissues are not well understood. These are controlled in human physiology by a complex interplay of alloantigen distribution and presentation, cellular/humoral active and passive tolerance, hormones/chemokines/angiogenic factors and their gradients, systemic and local microbial communities. Reductive discrimination of these factors in physiology and pathology of model systems and humans requires simplification of the model and increased resolution of interrogative technologies. As a baseline, we have studied the gestational tissue dynamics in the syngeneic C57BL/6 mice, as the simplest immunological environment, and focused on validating the approach to increased data density and computational analysis pipeline afforded by highly polychromatic flow cytometry and machine learning interpretation. We mapped DC and T cell subsets, and comprehensively examined their maternal (decidual)—fetal (placental) interface dynamics. Both frequency and composition of decidual DCs changed across gestation, with a dramatic increase in myeloid DCs in early pregnancy, and exclusion of plasmacytoid DCs. CD4+ T cells, in contrast, were lower at all gestational ages and an unusual CD4−CD8−TCRαβ+group was prominent at mid-pregnancy. Dimensionality reduction with machine learning-aided clustering revealed that CD4−CD8− T cells were phenotypically different from CD4+ and CD8+ T cells. Additionally, divergence between maternal decidual and fetal placental compartment was prominent, with absence of DCs from the placenta, but not decidua or embryo. These results provide a novel framework and a syngeneic baseline on which the specific role of alloantigen/tolerance, polymicrobial environment, and models of pregnancy pathology can be precisely modeled and analyzed.

Cyclic Nucleotides Differentially Regulate Cx43 Gap Junction Function in Uterine Artery Endothelial Cells From Pregnant Ewes

Cell–cell communication is dependent on GJ (gap junction) proteins such as Cx43 (connexin 43). We previously demonstrated the importance of Cx43 function in establishing the enhanced pregnancy vasodilatory phenotype during pregnancy in uterine artery endothelial cells from pregnant (P-UAEC) ewes. Cx43 is regulated by elevating cAMP and PKA (protein kinase A)–dependent Cx43 S365 phosphorylation–associated trafficking and GJ open gating, which is opposed by PKC (protein kinase C)–dependent S368 phosphorylation-mediated GJ turnover and closed gating. However, the role of cyclic nucleotide-mediated signaling mechanisms that control Cx43 and GJ function in P-UAECs is unknown. We hypothesize that cAMP will mediate increases in S365 phosphorylation, thereby, enhancing GJ trafficking and open gating, while cGMP will stimulate S368, but not S365, phosphorylation to enhance GJ turnover and closed gating in P-UAECs. Treatment with 8-Bromo (8-Br)-cAMP signal significantly (P<0.05) increased nonphosphorylated S365 signal and total Cx43 phosphorylation, but not S368 phosphorylation, while 8-Br-cGMP significantly (P<0.05) increased Cx43 C-terminus-S365 signal, S368, and total Cx43 phosphorylation. Inhibition of PKA, but not PKG (protein kinase G), abrogated the 8-Br-cAMP–stimulated increase in nonphosphorylated S365 and total Cx43 phosphorylation and inhibited S368 below basal levels, whereas inhibition of PKG blocked (P<0.05) the 8-bromo-cGMP-stimulated rises in nonphosphorylated S365, total Cx43, and S368 phosphorylation levels in P-UAECs. Functional studies showed that 8-Br-cAMP increased dye transfer and sustained calcium bursts, while 8-Br-cGMP decreased both. Thus, in P-UAECs, only 8-Br-cAMP and not 8-Br-cGMP effectively enhances nonphosphorylated S365 and total Cx43 expression that correspondingly reduces S368 phosphorylation, allowing increased GJ communication. This provides new insights into the regulatory mechanisms behind Cx43 function and GJ communication.

Uterine artery leptin receptors during the ovarian cycle and pregnancy regulate angiogenesis in ovine uterine artery endothelial cells.

Leptin regulates body weight, reproductive functions, blood pressure, endothelial function, and fetoplacental angiogenesis. Compared to the luteal phase, the follicular phase and pregnancy are physiological states of elevated estrogen, angiogenesis, and uterine blood flow (UBF). Little is known concerning regulation of uterine artery (UA) angiogenesis by leptin and its receptors. We hypothesized that (1) ex vivo expression of leptin receptors (LEPR) in UA endothelium (UAendo) and UA vascular smooth muscle (UAvsm) is elevated in pregnant versus nonpregnant (Luteal and Follicular) sheep; (2) in vitro leptin treatments differentially modulate mitogenesis in uterine artery endothelial cells from pregnant (P-UAECs) more than in nonpregnant (NP-UAECs) ewes; and (3) LEPR are upregulated in P-UAECs versus NP-UAECs in association with leptin activation of phospho-STAT3 signaling. Local UA adaptations were evaluated using a unilateral pregnant sheep modelwhere prebreeding uterine horn isolation (nongravid) restricted gravidity to one horn. Immunolocalization revealed LEPR in UAendo and UAvsm from pregnant and nonpregnant sheep. Contrary to our hypothesis, western analysis revealed that follicular UAendo and UAvsm LEPR were greater than luteal, nongravid, gravid, and control pregnant. Compared to pregnant groups, LEPR were elevated in renal artery endothelium of follicular and luteal sheep. Leptin treatment significantly increased mitogenesis in follicular phase NP-UAECs and P-UAECs, but not luteal phase NP-UAECs. Although UAEC expression of LEPR was similar between groups, leptin treatment only activated phospho-STAT3 in follicular NP-UAECs and P-UAECs. Thus, leptin may play an angiogenic role particularly in preparation for the increased UBF during the periovulatory period and subsequently to meet the demands of the growing fetus. Summary Sentence Leptin receptors are locally expressed in uterine artery endothelium and vascular smooth muscle cells during the ovarian cycle and late pregnancy. Leptin may play a role in regulating angiogenic responses of uterine artery endothelial cells during the follicular phase and late pregnancy.

Regulación del flujo sanguíneo uterino: I. Funciones de estrógeno y receptores estrogénicos α/β en el endotelio vascular uterino durante el embarazo

Estrogen and classical estrogen receptors (ERs), ER-α and ER-β, have been shown to be partially responsible for short and long term uterine endothelial adaptations during pregnancy. The molecular and structural differences, together with the various effects caused by these receptors in cells and tissues, suggest that their function varies depending upon estrogen and estrogen receptor signaling. In this review, we discuss the role of estrogen and its classic receptors in the cardiovascular adaptations during pregnancy and the expression of ERs in vivo and in vitro in the uterine artery endothelium during the ovarian cycle and pregnancy, while comparing their expression in arterial endothelium from reproductive and non-reproductive tissues. These themes integrate current knowledge of this broad scientific field with various interpretations and hypothesis that related estrogenic effects by either one or both ERs. This review also includes the relationship with vasodilator and angiogenic adaptations required to modulate the dramatic physiological increase to the uteroplacental perfusion observed during normal pregnancy.

Regulación del flujo sanguíneo uterino: II. Funciones de estrógeno y receptores estrogénicos α/β en acciones genómicas y no-genómicas del endotelio uterino

El embarazo esta marcado por cambios y adaptaciones cardiovasculares que son importantes para el crecimiento y mantenimiento de la placenta y el feto. Durante este periodo, las adaptaciones vasculares uterinas manifiestan cambios clasificados como de corto o largo plazo los cuales estan relacionados con adaptaciones vasodilatadoras, angiogenicas o de remodelacion. El estrogeno y los receptores estrogenicos clasicos (REs), RE-α y RE-β, han demostrado ser parcialmente responsables por facilitar el incremento dramatico en el fluido sanguineo uterino necesario durante el embarazo. En esta revision bibliografica se discuten la base estructural para la diversidad y selectividad funcional de los REs por el estrogeno, el papel de los REs sobre los efectos genomicos y no-genomicos en celulas endoteliales de arterias uterinas (CEAU). Estos temas integran el conocimiento cientifico sobre la regulacion molecular de CEAU para mantener el incremento fisiologico en la perfusion utero-placentaria observada durante un embarazo normal.