Thomas J. Lechuga

Progesterone withdrawal promotes remodeling processes in the nonpregnant mouse cervix.

Abstract Prepartum cervical ripening is associated with remodeling of collagen structure and with inflammation. Progesterone withdrawal is critical for parturition, but the effects of progesterone decline on cervical morphology are unknown. The present study tested the hypothesis that progesterone withdrawal promotes processes associated with remodeling of the cervix. Adult, virgin, female C57BL/6 mice received silastic capsules with oil vehicle or estradiol plus progesterone to parallel concentrations in circulation during pregnancy. After 17 days of estradiol and progesterone treatment, the progesterone implant was removed from one group. Mice in each group were killed 15, 18, or 19 days after placement of capsules. Sections of cervix were stained for collagen, and the densities of macrophages, neutrophils, and area with nerve fibers were assessed. Treatment with gonadal steroids promoted hypertrophy of the cervix, as well as reduced collagen and increased area with nerve fibers compared with vehicle-treated controls. Removal of the progesterone capsule did not affect hypertrophy or innervation, but it did reduce collagen. By contrast, significantly more macrophages and neutrophils were present in the cervix on Days 18 and 19 (i.e., by 24 and 48 h after withdrawal of the progesterone capsule); the immune cell census was equivalent to that in vehicle controls. Findings indicate that gonadal steroids, comparable to those during pregnancy, promote hypertrophy and suppress immigration of immune cells in the cervix. Therefore, in a nonpregnant murine model for parturition, progesterone withdrawal is suggested to recruit immune cells and processes that remodel the cervix.

Transection of the Pelvic or Vagus Nerve Forestalls Ripening of the Cervix and Delays Birth in Rats

Innervation of the cervix is important for normal timing of birth because transection of the pelvic nerve forestalls birth and causes dystocia. To discover whether transection of the parasympathetic innervation of the cervix affects cervical ripening in the process of parturition was the objective of the present study. Rats on Day 16 of pregnancy had the pelvic nerve (PnX) or the vagus nerve (VnX) or both pathways (PnX+VnX) transected, sham-operated (Sham) or nonpregnant rats served as controls. Sections of fixed peripartum cervix were stained for collagen or processed by immunohistochemistry to identify macrophages and nerve fibers. All Sham controls delivered by the morning of Day 22 postbreeding, while births were delayed in more than 75% of neurectomized rats by more than 12 h. Dystocia was evident in more than 25% of the PnX and PnX+VnX rats. Moreover, on prepartum Day 21, serum progesterone was increased severalfold in neurectomized versus Sham rats. Assessments of cell nuclei counts indicated that the cervix of neurectomized rats and Sham controls had become equally hypertrophied compared to the unripe cervix in nonpregnant rats. Collagen content and structure were reduced in the cervix of all pregnant rats, whether neurectomized or Shams, versus that in nonpregnant rats. Stereological analysis of cervix sections found reduced numbers of resident macrophages in prepartum PnX and PnX+VnX rats on Day 21 postbreeding, as well as in VnX rats on Day 22 postbreeding compared to that in Sham controls. Finally, nerve transections blocked the prepartum increase in innervation that occurred in Sham rats on Day 21 postbreeding. These findings indicate that parasympathetic innervation of the cervix mediates local inflammatory processes, withdrawal of progesterone in circulation, and the normal timing of birth. Therefore, pelvic and vagal nerves regulate macrophage immigration and nerve fiber density but may not be involved in final remodeling of the extracellular matrix in the prepartum cervix. These findings support the contention that immigration of immune cells and enhanced innervation are involved in processes that remodel the cervix and time parturition.

Remodeling of the Cervix and Parturition in Mice Lacking the Progesterone Receptor B Isoform

Withdrawal of progestational support for pregnancy is part of the final common pathways for parturition, but the role of nuclear progesterone receptor (PGR) isoforms in this process is not known. To determine if the PGR-B isoform participates in cervical remodeling at term, cervices were obtained from mice lacking PGR-B (PGR-BKO) and from wild-type (WT) controls before or after birth. PGR-BKO mice gave birth to viable pups at the same time as WT controls during the early morning of Day 19 postbreeding. Morphological analyses indicated that by the day before birth, cervices from PGR-BKO and WT mice had increased in size, with fewer cell nuclei/area as well as diminished collagen content and structure, as evidenced by optical density of picrosirius red-stained sections, compared to cervices from nonpregnant mice. Moreover, increased numbers of resident macrophages, but not neutrophils, were found in the prepartum cervix of PGR-BKO compared to nonpregnant mice, parallel to findings in WT mice. These results suggest that PGR-B does not contribute to the growth or degradation of the extracellular matrix or proinflammatory processes associated with recruitment of macrophages in the cervix leading up to birth. Rather, other receptors may contribute to the progesterone-dependent mechanism that promotes remodeling of the cervix during pregnancy and in the proinflammatory process associated with ripening before parturition. A progesterone-mediated receptor mechanism that does not involve the progesterone receptor-B isoform maintains pregnancy and regulates cervical remodeling and parturition.

Estrogen Replacement Therapy in Ovariectomized Nonpregnant Ewes Stimulates Uterine Artery Hydrogen Sulfide Biosynthesis by Selectively Up-Regulating Cystathionine β-Synthase Expression

Estrogens dramatically dilate numerous vascular beds with the greatest response in the uterus. Endogenous hydrogen sulfide (H2S) is a potent vasodilator and proangiogenic second messenger, which is synthesized from L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). We hypothesized that estrogen replacement therapy (ERT) selectively stimulates H2S biosynthesis in uterine artery (UA) and other systemic arteries. Intact and endothelium-denuded UA, mesenteric artery (MA), and carotid artery (CA) were obtained from ovariectomized nonpregnant ewes (n = 5/group) receiving vehicle or estradiol-17β replacement therapy (ERT). Total RNA and protein were extracted for measuring CBS and CSE, and H2S production was determined by the methylene blue assay. Paraffin-embedded UA rings were used to localize CBS and CSE proteins by immunofluorescence microscopy. ERT significantly stimulated CBS mRNA and protein without altering CSE mRNA or protein in intact and denuded UA. Quantitative immunofluorescence microscopic analyses showed CBS and CSE protein localization in endothelium and smooth muscle and confirmed that ERT stimulated CBS but not CSE protein expression in UA endothelium and smooth muscle. ERT also stimulated CBS, but not CSE, mRNA and protein expression in intact and denuded MA but not CA in ovariectomized ewes. Concomitantly, ERT stimulated UA and MA but not CA H2S production. ERT-stimulated UA H2S production was completely blocked by a specific CBS but not CSE inhibitor. Thus, ERT selectively stimulates UA and MA but not CA H2S biosynthesis by specifically up-regulating CBS expression, implicating a role of H2S in estrogen-induced vasodilation and postmenopausal womens health.

Cervix remodeling and parturition in the rat: lack of a role for hypogastric innervation

The hypogastric nerve is a major pathway innervating the uterine cervix, yet its contribution to the processes of cervical ripening and parturition is not known. The main objective of this study was to determine the effect of hypogastric nerve transection on remodeling of the cervix and timing of birth. As an initial goal, processes associated with remodeling of the peripartum cervix were studied. The cervix was obtained from time-dated pregnant rats on days 15, 19, 21, and 21.5 of pregnancy, and post partum on the day of birth (day 22). The cervix was excised, post-fixed overnight, and sections stained to evaluate collagen content and structure or processed by immunohistochemistry to identify macrophages or nerve fibers. The census of macrophages and density of nerve fibers in the cervix peaked on day 21, the day before birth, and then declined post partum. These results replicate in time course and magnitude previous studies in mice. To address the main objective, the hypogastric nerve was bilaterally transected on day 15 post-breeding; sham-operated rats served as controls. Pups were born in both groups at normal term. Transection of the hypogastric nerves did not affect remodeling of collagen or the census of macrophages or the density of nerve fibers in the cervix. These findings support the contention that enhanced innervation and immigration of immune cells are associated with remodeling of the cervix and parturition, but that a neural pathway other than the hypogastric nerve may participate in the process of cervical ripening.

S-Nitrosylation of Cofilin-1 Mediates Estradiol-17β-Stimulated Endothelial Cytoskeleton Remodeling

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.

Augmented H2S production via cystathionine-beta-synthase upregulation plays a role in pregnancy-associated uterine vasodilation

Abstract Endogenous hydrogen sulfide (H2S) synthesized via metabolizing L-cysteine by cystathionine-betasynthase (CBS) and cystathionine-gamma-lyase (CSE) is a potent vasodilator and angiogenic factor. The objectives of this study were to determine if human uterine artery (UA) H2S production increases with augmented expression and/or activity of CBS and/or CSE during the menstrual cycle and pregnancy and whether exogenous H2S dilates UA. Uterine arteries from nonpregnant (NP) premenopausal proliferative (pPRM) and secretory (sPRM) phases of the menstrual cycle and pregnant (P) women were studied. H2S production was measured by the methylene blue assay. CBS and CSE mRNAs were assessed by quantitative real-time PCR, and proteins were assessed by immunoblotting and semiquantitative immunofluorescence microscopy. Effects of H2S on rat UA relaxation were determined by wire myography ex vivo. H2S production was greater in NP pPRM and P than NP sPRM UAs and inhibited by the specific CBS but not CSE inhibitor. CBS but not CSE mRNA and protein were greater in NP pPRM and P than NP sPRM UAs. CBS protein was localized to endothelium and smooth muscle and its levels were in a quantitative order of P >NP UAs of pPRM>sPRM. CSE protein was localized in UA endothelium and smooth muscle with no difference among groups. A H2S donor relaxed P > NP UAs but not mesentery artery. Thus, human UA H2S production is augmented with endothelium and smooth muscle CBS upregulation, contributing to UA vasodilation in the estrogen-dominant physiological states in the proliferative phase of the menstrual cycle and pregnancy. Summary Sentence Augmented hydrogen sulfide biosynthesis via upregulating endothelium and smooth muscle cystathionine β-synthase expression plays a role in pregnancy-associated uterine vasodilation.

Pregnancy-related changes in connections from the cervix to forebrain and hypothalamus in mice.

The transneuronal tracer pseudorabies virus was used to test the hypothesis that connections from the cervix to the forebrain and hypothalamus are maintained with pregnancy. The virus was injected into the cervix of nonpregnant or pregnant mice, and, after 5 days, virus-labeled cells and fibers were found in specific forebrain regions and, most prominently, in portions of the hypothalamic paraventricular nucleus. With pregnancy, fewer neurons and fibers were evident in most brain regions compared to that in nonpregnant mice. In particular, little or no virus was found in the medial and ventral parvocellular subdivisions, anteroventral periventricular nucleus, or motor cortex in pregnant mice. By contrast, labeling of virus was sustained in the dorsal hypothalamus and suprachiasmatic nucleus in all groups. Based upon image analysis of digitized photomicrographs, the area with label in the rostral and medial parvocellular paraventricular nucleus and magnocellular subdivisions was significantly reduced in mice whose cervix was injected with virus during pregnancy than in nonpregnant mice. The findings indicate that connections from the cervix to brain regions that are involved in sensory input and integrative autonomic functions are reduced during pregnancy. The findings raise the possibility that remaining pathways from the cervix to the forebrain and hypothalamus may be important for control of pituitary neuroendocrine secretion, as well as for effector functions in the cervix as pregnancy nears term.

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

ABSTRACT 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-12C614N4-Arg and L-12C614N2-Lys) or “heavy” (L-13C615N4-Arg and L-13C615N2-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.

Estradiol-17β stimulates H2S biosynthesis by ER-dependent CBS and CSE transcription in uterine artery smooth muscle cells in vitro: LECHUGA et al.

Endogenous hydrogen sulfide (H2S), synthesized by cystathionine β‐synthase (CBS) and cystathionine γ‐lyase (CSE), is a potent vasodilator that can be stimulated by estradiol‐17β (E 2β) in uterine artery (UA) smooth muscle (UASMC) in vivo; however, the underlying mechanisms are unknown. This study tested a hypothesis that E 2β stimulates H 2S biosynthesis by upregulating CBS expression via specific estrogen receptor (ER). Treatment with E 2β stimulated time‐ and concentration‐ dependent CBS and CSE messenger RNA (mRNA) and protein expressions, and H 2S 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 2β‐stimulated responses, which were blocked by specific ERα or ERβ antagonist. Moreover, E 2β activated both CBS and CSE promoters and ICI 182,780 blocked the E 2β‐stimulated responses. Thus, E 2β stimulates H 2S production by upregulating CBS and CSE expression via specific ER‐dependent transcription in UASMC in vitro.