Regulation Of Uterine Blood Flow Research Articles

Maternal B cell signaling orchestrates fetal development in mice.

B cell participation in early embryo/fetal development and the underlying molecular pathways have not been explored. To understand whether maternal B cell absence or impaired signaling interferes with placental and fetal growth, we paired CD19-deficient (CD19-/-) mice, females with B cell-specific MyD88 (BMyD88-/-) or IL10 (BIL10-/-) deficiency as well as wild-type and MyD88-/- controls on C57Bl/6 background with BALB/c males. Pregnancies were followed by ultrasound and Doppler measurements. Implantation number was reduced in BMyD88-/- and MyD88-/- mice. Loss of MyD88 or B cell-specific deletion of MyD88 or IL10 resulted in decreased implantation areas at gestational day (gd) 5, gd8 and gd10, accompanied by reduced placental thickness, diameter and areas at gd10. Uterine artery resistance was enhanced in BIL10-/- dams at gd10. Challenge with 0.4 mg lipopolysaccharide/kg bodyweight at gd16 revealed that BMyD88-/-, BIL10-/- and CD19-/- mothers delivered preterm, whereas controls maintained their pregnancy. B cell-specific MyD88 and IL10 expression is essential for appropriate in utero development. IL10+B cells are involved in uterine blood flow regulation during pregnancy. Finally, B cell-specific CD19, MyD88 and IL10 expression influences susceptibility towards preterm birth.

Cyclooxygenase-dependent mechanisms mediate in part the anti-dilatory effects of perivascular adipose tissue in uterine arteries from pregnant rats

Uterine perivascular adipose tissue (PVAT) contributes to uterine blood flow regulation in pregnancy, at least in part, due to its effects on uterine artery reactivity. We tested the hypothesis that uterine PVAT modulates the balance between the contribution of nitric oxide synthase (NOS)- and cyclooxygenase (COX)-dependent pathways to acetylcholine (ACh)-induced relaxation in isolated uterine arteries. Concentration-response curves to ACh (1 nM – 30 µM) were performed on uterine arteries from pregnant and non-pregnant rats. Arteries were exposed to Krebs-Henseleit solution (control) or PVAT-conditioned media (PVATmedia) in the presence of the following inhibitors: L-NAME (NOS inhibitor), indomethacin (COX inhibitor), SC560 (COX-1 inhibitor), NS398 (COX-2 inhibitor), SQ 29,548 (thromboxane receptor (TP) inhibitor). In arteries incubated with PVATmedia, the presence of indomethacin increased ACh-induced relaxation, reversing the anti-dilatory effect of PVATmedia. NOS inhibition reduced ACh-induced relaxation in uterine arteries from pregnant rats, and exposure to PVATmedia did not change this effect. Selective inhibition of COX-1 but not COX-2 suppressed relaxation responses to ACh in control arteries. The presence of PVATmedia abolished the effect of COX-1 inhibition. Incubation of uterine arteries from pregnant rats with PVATmedia increased production of thromboxane B2 (TxB2, p = 0.01) but thromboxane receptor (TP) inhibition did not affect the anti-dilatory properties of PVATmedia. In conclusion, inhibition of COX signaling suppressed the anti-dilatory effects of PVATmedia, while PVATmedia had no effect on the contribution of the NOS/NO pathway to ACh-induced relaxation in uterine arteries from pregnant rats, indicating that the anti-dilatory effects of uterine PVAT are mediated in part by COX-dependent mechanisms.

Abstract P224: Cyclooxygenase-dependent Mechanisms Mediate In Part The Anti-dilatory Effects Of Uterine Perivascular Adipose Tissue In Rat Pregnancy

Introduction: Uterine perivascular adipose tissue (PVAT) contributes to uterine blood flow regulation in pregnancy, at least in part, due to its effects on uterine artery tone. We hypothesized that the anti-dilatory effects of uterine PVAT are mediated by vascular nitric oxide synthase (NOS)- and cyclooxygenase (COX)-dependent mechanisms. Methods: Main uterine arteries from pregnant and non-pregnant rats were mounted onto a wire myograph. Concentration-response curves to acetylcholine (ACh, 10 -9 - 3x10 -5 M) were performed on arteries exposed to physiological salt solution or PVAT-conditioned media (PVAT media ) in the presence of the following inhibitors: a) L-NAME (NOS inhibitor, 100 μM), b) indomethacin (COX inhibitor, 10 μM), c) SC560 (COX-1 inhibitor, 1 μM), d) NS398 (COX-2 inhibitor, 1 μM)]. Results: NOS inhibition abolished ACh-induced relaxation in uterine arteries from pregnant rats and exposure to PVAT media did not change this effect [AUC, (-)PVAT media : 244.6 ± 18.1 vs. (-)PVAT media /(+)L-NAME: 52.64 ± 7.4, p < 0.0001; (+)PVAT media : 202.4 ± 15.5 vs. (+)PVAT media /(+)L-NAME: 56.17 ± 11.3, p < 0.0001]. Indomethacin suppressed ACh-induced relaxation in uterine arteries from pregnant rats [AUC, (-)PVAT media : 243.6 ± 6.6 vs. (-)PVAT media /(+)Indomethacin: 123.6 ± 12.3, p < 0.0001] but not in non-pregnant rats (p>1.0). In arteries incubated with PVAT media , the presence of indomethacin increased ACh-induced relaxation [AUC, (+)PVAT media : 125.2 ± 11.4 vs. (+)PVAT media /(+)Indomethacin: 179.1 ± 14.7, p = 0.01]. COX-1 but not COX-2 inhibition suppressed relaxation responses to ACh [AUC, COX-1 inhibition, (-)PVAT media : 244.6 ± 12.0 vs. (-)PVAT media /(+)SC560: 142.4 ± 15.4, p = 0.02; COX-2 inhibition, (-)PVAT media vs. (-)PVAT media /(+)NS398, p=0.1). The anti-dilatory effects of PVAT were not observed in the presence of SC560 or NS398 (p>0.05). Exposure to PVAT media increased the protein content of COX-1 (p=0.05) and COX-2 (p=0.03) and the production of thromboxane B 2 (p=0.01) in uterine arteries from pregnant rats. Conclusion: The anti-dilatory effects of PVAT-derived factors on uterine arteries are mediated in part by COX-derived products and this mechanism is specific to pregnancy.

Exosome‐like Vesicles Facilitate Intercellular Communication between Uterine Artery Smooth Muscle Cells and Perivascular Adipose Tissue

IntroductionPerivascular adipose tissue (PVAT) contributes to regulation of vascular tone. We recently reported that PVAT surrounding uterine arteries had anti‐dilatory effects on isolated uterine arteries and contributed to local regulation of uterine blood flow in pregnant rats. The objective of the current study was to investigate the mechanisms underlying the functional interaction between PVAT and uterine arterial wall. Adipose tissue passively secretes exosomes, which are membrane bound vesicles (30 – 150 nm in diameter). It is unknown whether PVAT, which has a different developmental origin from other adipose depots, secretes exosomes, and if exosomes facilitate intercellular communication between PVAT and vascular cells. We hypothesized that uterine PVAT sheds exosome‐like vesicles (Exo‐PVAT) that transfer their cargo into neighboring uterine vascular smooth muscle cells (USMCs), facilitating maternal uterine artery function.MethodsExo‐PVAT and primary USMCs were isolated from pregnant (gestational day 16, term=22–23 days) and non‐pregnant rats. Exosomes were isolated and purified with tissue culture, and ultrafiltration and ultracentrifugation techniques, and USMCs were isolated using enzymatic digestion. Exosome morphology was characterized via transmission electron microscopy, and exosome size distribution and molecular weight were determined via a Malvern Zetasizer and fast protein liquid chromatography (FPLC), respectively. Protein expression of various exosome markers was determined using Western blot analysis. To determine USMC uptake of Exo‐PVAT, uterine Exo‐PVAT were labelled with a membrane‐labeling dye (DiO, Invitrogen) and then co‐cultured with USMCs for 3 hours. Vascular reactivity to acetylcholine (ACh; 10−9 – 3×10−5 M) was measured in uterine arteries using wire myography after exposure to Exo‐PVAT or phosphate‐buffered saline (PBS, control) for 3 hours.ResultsExo‐PVAT expressed TSG101, Alix, and CD9. Pregnancy did not affect Exo‐PVAT size [Median(IQR) (nm), Non‐pregnant: 99.4 (80.5) vs. Pregnant: 46.7 (21.2), p=0.5]. Using FPLC, we identified exosomes of 40–200 kDa in samples from both pregnant and non‐pregnant rats. Samples from pregnant rats had a relatively high expression of exosomes of 2000 kDa when compared to samples from non‐pregnant rats. Using immunocytochemistry, we demonstrated that USMCs were able to take up exosomes derived from their adjacent PVAT. Using a functional bioassay, we found that Exo‐PVAT reduced sensitivity to ACh in uterine arteries from pregnant rats (pEC50, control: 7.46 vs. Exo‐PVATPREG: 7.02) but had the opposite effect in non‐pregnant rats (pEC50, control: 6.91 ± 0.04 vs. Exo‐PVATNP: 7.03 ± 0.06, p<0.01).ConclusionUterine PVAT sheds exosome‐like vesicles that transfer their cargo to the adjascent uterine arterial wall, where they are taken up by USMCs. The interaction between Exo‐PVAT and USMCs is a novel type of intercellular communication that may have important implications in uterine artery function and local regulation of uterine blood flow in pregnancy.Support or Funding InformationUNTHSC Seed Grant

In Remembrance of Thomas F. Ferris, MD, Former ASN President (1987–1988)

In Remembrance of Thomas F. Ferris, MD, Former ASN President (1987–1988)

Endocrine changes, fetal growth, and uterine artery hemodynamics after chronic estrogen suppression during the last trimester of equine pregnancy.

Equine pregnancy is characterized by very high circulating concentrations of estrogens. The physiological roles of estrogens during equine gestation are largely unknown, although some studies suggest a role in the regulation of uterine artery hemodynamics and a relationship between low circulating estrogen concentrations and late pregnancy loss. The objectives of this experiment were to evaluate the effects of estrogen suppression on uterine artery hemodynamics and on pregnancy outcome. Estrogen synthesis was suppressed using letrozole, a potent aromatase inhibitor. Twelve pregnant mares were randomly assigned to a control (n = 6) or treatment (n = 6; 500 mg letrozole orally every 4 days) group with treatment starting at 240 days of gestation and continuing until parturition. Weekly serum samples were analyzed to determine testosterone, dehydroepiandrosterone sulfate, estradiol, estrone sulfate, progestins, and prostaglandin F2α metabolite concentrations. Ultrasonographic examinations were performed biweekly and measurements included uterine artery hemodynamics (diameter, pulsatility, and resistance indices), fetal growth using the diameter of the fetal eye, and placental evaluation using the combined thickness of the uterus and placenta. At parturition, gestational length, foal weight, and neonatal viability were determined. Letrozole suppressed estrogen synthesis during gestation by approximately 90% compared to control values. This large reduction in circulating estrogens had no effect on uterine artery hemodynamics, normal placental development, maintenance of pregnancy, or neonatal viability. However, neonates from letrozole-treated mares had lower (P < 0.05) birth weights than controls, suggesting that estrogens may play a role in fetal growth that is not mediated through regulation of uterine blood flow.

Regulation of uterine blood flow. I. Functions of estrogen and estrogen receptor α/β in the uterine vascular endothelium during pregnancy

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.

Relationships Between Uterine Blood Flow, Peripheral Sex Steroids, Expression of Endometrial Estrogen Receptors and Nitric Oxide Synthases During the Estrous Cycle in Mares

The objective of this study was to investigate the relationships between uterine perfusion and estrogen, progesterone and the uterine nitric oxide synthase (NOS) system in five trotter mares during the estrous cycle. Color Doppler sonography for measurement of uterine blood flow and collection of blood for determination of plasma estrogen and progesterone concentrations were performed on days 0 (= ovulation), 1, 5, 11 and 15 and daily during estrus (days -1 to -4) of one estrous cycle; endometrial biopsy collection for mRNA expression analysis of NOS and estrogen receptors was performed on days 0, 1, 5, 11, 15 and -3. Blood flow in each uterine artery was assessed by calculating the mean time-averaged maximum velocity (TAMV) and the pulsatility index (PI). Plasma concentrations of estrogen and progesterone were determined using specific enzyme immunoassays. The mRNA expressions of endothelial NOS (eNOS), inducible NOS (iNOS) as well as estrogen receptors α (ERα) and β (ERβ) were quantified using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The TAMV and PI had a biphasic pattern during the estrous cycle (P<0.05), with maximum and minimum, respectively, values on days 5 and -4. Estrogen receptor mRNA concentrations increased significantly during days 15 (ERα) and -3 (ERβ). Transcript expression of eNOS, but not iNOS, had a biphasic pattern during the cycle (P<0.05) with maximum levels on days 5 and -3 and correlated positively with TAMV (r=0.81, P=0.05). We infer that the uterine NOS system, especially eNOS, plays an important role in the regulation of uterine blood flow during the estrous cycle in mares.

Estrogen Induced Uterine Artery Endothelial Nitrosyl-Proteome: Potential Roles of Protein Nitrosylation in Uterine Blood Flow Regulation.

Estrogen is one of the major driving forces for dilating uterine arteries to facilitate the dramatic rises in uterine blood flow that parallels the exponential growth phase of fetal weight during late gestation. Estrogen-induced and pregnancy-associated uterine vasodilatations occur via enhanced endothelial nitric oxide (NO) synthase (eNOS) expression/activation thereby increasing NO production locally at the level of the uterine artery. However, it is unknown whether increased NO production by estrogen stimulation directly affects proteins and their functions in uterine arteries. Formation of S-nitrothiols via addition of 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 with biological significance analogous to phosphorylation. To determine the effects of exogenous estrogen and NO treatments on protein nitrosylation and to profile the estrogen-induced nitrosyl-proteome in uterine artery endothelial cells. Primary cultured sheep uterine artery endothelial cells were treated with or without etradiol-17β (E2β, 10 nM) or a NO donor S-nitrosoglutathione (GSNO, 1 mM) for 30 min. Protein nitrosylation was measured by a modified 3-step biotin switch technique including blocking free thiols in the nitrosyl-proteomes of total protein samples by methylmethanethiosulfonate, followed by ascorbate reduction and labeling with the CyDye fluorescence tags. E2β or GSNO treated samples (Cy5 labeled, red) were mixed with equal amounts of control sample (Cy3 labeled, green) and then analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). High resolution digitalized fluorescence images of the 2D-DIGE were captured and changes in densities of nitrosyl-proteins by E2α or GSNO were quantified off-line. Protein spots of interest (based on increases or decreases in intensity) on the 2D-DIGE were picked up and identified by matrix-assisted laser desorption/ionization-time of flight (MALDI/TOF) mass spectrometry. There were >1000 nitrosylated proteins identified on 2D-DIGE. Among those, at least 25 proteins were significantly stimulated by E2β or GSNO. Interestingly, many proteins were also unexpectedly denitrosylated by estrogen or GSNO. With MALDI-TOF mass spectrometry we sequenced 100 nitrosyl-proteins of interest. We found that E2β and GSNO stimulated significantly different nitrosyl-proteomes. Some proteins were nitrosylated or denitrosylated by both E2β and GSNO, but others were only affected by one or the other. Pathway analysis of the identified nitrosyl-proteins suggested that protein nitrosylation is clearly involved in estrogen regulation of protein translation and degradation, mitochondrial redox balance, cell structure and metabolism, and ion homeostasis, etc. Our data showing estrogen stimulation of protein nitrosylation in uterine artery endothelial cells signifies a significant next step for the understanding of the biological targets of enhanced NO production by estrogen in uterine arteries that are associated with the regulation of uterine blood flow. The physiological consequences of nitrosylated/denitrosylated proteins by estrogen in uterine artery endothelial cells warrants for further investigation (NIH HL70562, HL74947 HL49210, HD38843, HL87144 and HL86939). (platform)

Mechanism of VEGF-Induced Uterine Venous Hyperpermeability

Background: Transfer of molecular signals from veins to adjacent arteries is an established mechanism of vascular communication in the uterine circulation, which ultimately depends on venous permeability. This study tests the hypotheses that: (1) uterine veins are permeable to intravenous solutes, using a 3-kDa dextran tracer and (2) this permeability is enhanced in response to vascular endothelial growth factor (VEGF). Additionally, the involvement of nitric oxide (NO), calcium, and the phospholipase C-protein kinase C (PLC-PKC) cascade in VEGF-enhanced permeability were investigated, and the impact of pregnancy-induced uterine vascular remodeling on permeability was evaluated. Methods: Studies utilized fluorimetry to quantitate solute flux in isolated segments of rat uterine vein as a function of endothelial surface area and time under basal and VEGF-stimulated conditions. VEGF signaling was probed using NO synthase inhibition (L-NNA), calcium channel blockade (lanthanum chloride) and withdrawal (calcium-free solution), and inhibitors of the PLC-PKC cascade (U-73122 and chelerythrine chloride, respectively). Gestational effects were assessed using vessels from late pregnant (day 20) rats. Results: Basal flux (control) in nonpregnant animals was 26 ± 2.5 molecules/µm²/min × 1,000 and was increased significantly by VEGF in a concentration-dependent manner (1 nM ≈ 3.3-fold, 10 nM ≈ 4.6-fold). Inhibition of PLC, PKC, and calcium signaling, but not NO, attenuated the response to VEGF. Gestation significantly increased flux (78 ± 9.3 molecules/µm²/min × 1,000), and maintained responsiveness to VEGF. Conclusions: These results demonstrate uterine venous permeability to intermediate-sized solutes through a VEGF-sensitive pathway involving calcium and PLC-PKC, but not NO, and further substantiate a role for veno-arterial communication in uterine blood flow regulation during pregnancy.

Induction of localized differences in rat uterine radial artery behavior and structure during gestation

Objectives The objectives of this study were to investigate differences in diameter and vasoconstriction of premyometrial versus uteroplacental radial arteries and to evaluate the contribution of nitric oxide (NO) to myogenic tone as a function of vessel location. Study design Radial arteries supplying either the myometrium or placenta were dissected from the uterus of pregnant rats. Constrictor responses to pressure elevation were studied before and after inhibition of endothelial NO synthase (eNOS). Results Passive lumen diameters of premyometrial and proximal uteroplacental arteries were comparable and significantly smaller than those of distal uteroplacental vessels. High potassium- and pressure-induced responses were also similar in premyometrial and proximal but were virtually absent in distal uteroplacental segments. L-NNA enhanced pressure-induced tone, but was without effect in distal uteroplacental segments. Conclusion Gestational remodeling alters arterial structure and reactivity in a highly localized manner. During pregnancy, enhanced basal production of NO may be an important local mechanism for uterine blood flow regulation.

Role of adenosine in regulation of uterine blood flow during nonpreeclamptic twin gestation.

We evaluated the possible role of increased adenosine in the regulation of uteroplacental circulation during nonpreeclamptic twin gestations. We performed uterine artery Doppler velocimetry and maternal arterial blood sampling for measurement of plasma adenosine levels in 16 women with singleton gestation and 14 women with dichorionic twin gestation without preeclampsia at 11-12, 25-26 and 34-36 weeks. In singleton gestations, a negative correlation between maternal plasma adenosine levels and maternal uterine artery pulsatility index was observed. In twin gestations, there were no noticeable association between maternal plasma adenosine levels and maternal uterine artery pulsatility index. Our results suggest that the decrease in vascular resistance in twin gestations may be through the mechanisms that are independent of adenosine.

Functional role of angiotensin II type 1 and 2 receptors in regulation of uterine blood flow in nonpregnant sheep.

The objective was to determine the receptor subtype of angiotensin II (ANG II) that is responsible for vasoconstriction in the nonpregnant ovine uterine and systemic vasculatures. Seven nonpregnant estrogenized ewes with indwelling uterine artery catheters and flow probes received bolus injections (0.1, 0.3 and 1 microg) of ANG II locally into the uterine artery followed by a systemic infusion of ANG II at 100 ng x kg(-1) x min(-1) for 10 min to determine uterine vasoconstrictor responses. Uterine ANG II dose-response curves were repeated following administration of the ANG II type 2 receptor (AT(2)) antagonist PD-123319 and then repeated again in the presence of an ANG II type 1 receptor (AT(1)) antagonist L-158809. In a second experiment, designed to investigate the mechanism of ANG II potentiation that occurred in the presence of AT(2) blockade, nonestrogenized sheep received a uterine artery infusion of L-158809 (3 mg/min for 5 min) prior to the infusion of 0.03 microg/min of ANG II for 10 min. ANG II produced dose-dependent decreases in uterine blood flow (P < 0.03), which were potentiated in the presence of the AT(2) antagonist (P < 0.02). Addition of the AT(1) antagonist abolished the uterine vascular responses and blocked ANG II-induced increases in systemic arterial pressure (P < 0.01). Significant uterine vasodilation (P < 0.01) was noted with AT(1) blockade in the second experiment, which was reversed by administration of the AT(2) antagonist or by the nitric oxide synthetase inhibitor N(omega)-nitro-L-arginine methyl ester. We conclude that the AT(1)-receptors mediate the systemic and uterine vasoconstrictor responses to ANG II in the nonpregnant ewe. AT(2)-receptor blockade resulted in a potentiation of the uterine vasoconstrictor response to ANG II, suggesting that the AT(2)-receptor subtype may modulate uterine vascular responses to ANG II potentially by release of nitric oxide.

Effects of the vasopressin V1a receptor antagonist, SR 49059, on the response of human uterine arteries to vasopressin and other vasoactive substances

Arginine vasopressin (AVP) activates the uterus via V1a receptors and is apparently an important factor for the myometrial hyperactivity, uterine ischemia and pain of primary dysmenorrhea. The orally active and selective, non-peptide AVP1a receptor antagonist, SR 49059, has been shown to inhibit the myometrial action of AVP, but the specific influence of this substance on the effects of AVP and other vasoactive agents on human uterine arteries is unknown. Concentration-responses of AVP on isolated medium-sized human uterine arteries were studied after incubation with only vehicle (DMSO, 0.1%) and with SR 49059 in concentrations of 0.5, 2.5 and 10 nmol/L. Furthermore, the concentration-responses of AVP were investigated without and with SR 49059 (2 and 10 nmol/L) on small and medium-sized arteries. Finally, the influence of 2.5 nmol/L of SR 49059 on concentration-responses of endothelin-1, noradrenaline and prostaglandin F2 alpha was studied. The EC50 for AVP on medium-sized arteries was 0.53 +/- 13 nmol/L. SR 49059 caused a competitive, dose-dependent inhibition of AVP-responses, the highest concentration giving an EC50 of 460 nmol/L. The pA2 value was 9.84. The responses of the small artery preparations to AVP, both without and with the antagonist, were more pronounced than those of the medium-sized ones. The vasoconstrictive effects of endothelin-1, noradrenaline and prostaglandin F2 alpha were less pronounced than those of AVP and unaffected by pre-exposure to SR 49059. The high potency of AVP on human uterine arteries, particularly those of small size, supports an involvement of the peptide in the regulation of uterine blood flow in both physiological and pathophysiological condition. SR 49059 is a potent and selective AVP V1a receptor antagonist in the smooth muscle of human uterine arteries.

Endothelin and neutral endopeptidase in the endometrium of women with menorrhagia.

The mechanisms underlying excessive menstrual bleeding or menorrhagia are not understood. In view of its potent vasoconstrictor and growth factor properties, endothelin has been proposed to have a potential paracrine role in the regulation of uterine blood flow and therefore could be a factor in menorrhagia. We compared the cellular localization of endothelin and its metabolizing enzyme, neutral endopeptidase, in endometrial biopsies from women with documented menorrhagia and in those with a normal menstrual cycle. Menorrhagia was documented by measurement of menstrual blood loss, 146 +/- 141 ml (median +/- SD). Endothelin and neutral endopeptidase were localized by immunohistochemistry, and the staining intensity was graded. Their immunostaining patterns were found to differ in menorrhagia compared to the normal menstrual cycle. Endothelin was reduced in glandular epithelium in menorrhagia and did not vary cyclically, while neutral endopeptidase was increased in the glandular epithelium. In menorrhagia, stromal endothelin immunoreactivity was not different from the normal cycle and although neutral endopeptidase immunostaining in stroma was similar to the secretory phase of normal endometrium, cyclical variation was absent. The potential for increased metabolism of endothelin could be an explanation for the decreased endothelin immunostaining in the glandular epithelium.

Evidence of interplay between plasma endothelin-1 and 17β-estradiol in regulation of uterine blood flow and endometrial growth in infertile women

Objective: To determine the effects of gonadotropin-induced ovarian stimulation on the plasma levels of endothelin-1 as well as on uterine blood flow and endometrial thickness. Design: Controlled cross-over clinical study. Setting: Academic research environment. Patient(s): Eighteen volunteer infertile women (31.4 ± 2.5 years, mean ± SE) participating in an IVF-ET program. Intervention(s): Transvaginal Doppler ultrasound was performed and blood samples were collected 4 days after the LH surge in a spontaneous cycle and 4 days after hCG administration in a subsequent gonadotropin-stimulated cycle. Main Outcome Measure(s): Plasma levels of E 2, P, and endothelin-1; uterine artery pulsatility index; and endometrial thickness. Result(s): Stimulated cycles were associated with significantly higher E 2 levels (101.0 ± 10.2 versus 723.5 ± 57.3 pg/mL [conversion Factor to SI unit, 3.671]; spontaneous versus stimulated), thicker endometrium (9.2 ± 2.5 versus 11.4 ± 2.9 mm), and lower endothelin-1 levels (2.5 ± 0.1 versus 2.1 ± 0.1 pg/mL [conversion factor to SI unit, 0.4]) and pulsatility index (3.22 ± 0.12 versus 2.63 ± 0.08) than spontaneous cycles. Endothelin-1 correlated negatively with E 2 (r = −0.38), but not with P or pulsatility index. Differences in endothelin-1 and endometrial thickness between spontaneous and stimulated cycles correlated negatively (r = −0.56) with each other. Conclusion: Our results suggest an inhibitory role for E 2 in the release of endothelin-1, which could be a factor in the regulation of uterine and endometrial blood flow.

Decidual renin secretion is modulated by endothelial cells

Because decidual renin may regulate uterine blood flow and decidual-endothelial cell proximity exists, we examined the modulation of decidual renin secretion by endothelial cells. Primary human decidual cell cultures were established. The effect of endothelial cells was studied by decidual-endothelial cocultures. Human umbilical endothelial cells were plated in half of the wells containing decidual cells and were then incubated for 24 h in serum-free media. The effect of endothelial cells was also studied by examining cAMP-mediated renin secretion using 10(-5) M forskolin under standardized conditions. We also examined the role of cell-to-cell contact by coculturing endothelial cells directly on decidual cultures and on cell inserts placed in decidual culture wells. The media were assayed for renin by radioimmunoassay (RIA) for angiotensin I (AI) after AI generation under standardized conditions. Statistical differences were analyzed by two-way analysis of variance (ANOVA) with randomized complete block design. Basal renin secretion, expressed as mean +/- SEM was 10.3 +/- 1.0 ng AI/ml/h. cAMP stimulation increased this to 15.5 +/- 1.3 ng AI/ml/h, P < 0.02. Coculture with vascular endothelium increased renin secretion to 24.3 +/- 2.4 ng AI/ml/h, P < 0.001. cAMP stimulation in cocultures increased renin secretion to 38.0 +/- 4.4 ng AI/ml/h, P < 0.002. There was no effect on cell growth as examined by total protein concentrations by coculturing or by cAMP. There was no increase in renin secretion when endothelial cells were cocultured using cell well inserts. Our data suggest that decidual renin secretion is increased by the presence of endothelial cells in direct contact with decidual cells. This supports a concept that in vivo-decidual-endothelial interaction may modulate decidual renin secretion and thereby influence regulation of uterine blood flow.

Decidual Renin Secretion is Modulated by Endothelial Cells

Because decidual renin may regulate uterine blood flow and decidual-endothelial cell proximity exists, we examined the modulation of decidual renin secretion by endothelial cells. Primary human decidual cell cultures were established. The effect of endothelial cells was studied by decidual-endothelial cocultures. Human umbilical endothelial cells were plated in half of the wells containing decidual cells and were then incubated for 24 h in serum-free media. The effect of endothelial cells was also studied by examining cAMP-mediated renin secretion using 105 M forskolin under standardized conditions. We also examined the role of cell-to-cell contact by coculturing endothelial cells directly on decidual cultures and on cell inserts placed in decidual culture wells. The media were assayed for renin by radioimmunoassay (RIA) for angiotensin I (AI) after AI generation under standardized conditions. Statistical differences were analyzed by two-way analysis of variance (ANOVA) with randomized complete block design. Basal renin secretion, expressed as mean ± SEM was 10.3 ± 1.0 ng AI/ml/h. cAMP stimulation increased this to 15.5 ± 1.3 ng AI/ml/h, P < 0.02. Coculture with vascular endothelium increased renin secretion to 24.3 ± 2.4 ng AI/ml/h, P < 0.001. cAMP stimulation in cocultures increased renin secretion to 38.0 ± 4–4 ng AI/ml/h, P < 0.002, There was no effect on cell growth as examined by total protein concentrations by coculturing or by cAMP. There was no increase in renin secretion when endothelial cells were cocultured using cell well inserts. Our data suggest that decidual renin secretion is increased by the presence of endothelial cells in direct contact with decidual cells. This supports a concept that in vivo-decidual-endothelial interaction may modulate decidual renin secretion and thereby influence regulation of uterine blood flow.

Immunolocalization of endothelin and neutral endopeptidase in the endometrium of users of subdermally implanted levonorgestrel (Norplant).

Subdermally implanted slow-release levonorgestrel (Norplant), a widely used effective contraceptive, has a high rate of discontinuation due to unacceptable menstrual bleeding disturbances. Endothelin (ET), a potent vasoconstrictor, varies across the menstrual cycle in normal endometrium. It has been proposed that ET has a potential paracrine role in the regulation of uterine blood flow. Neutral endopeptidase (NEP), a membrane-bound ecto-enzyme, can inactivate ET and is localized principally in endometrial stroma. We have compared the immuno-localization of ET and NEP in endometrial biopsies from Indonesian women using Norplant with normal controls. Differences were observed in the glandular and luminal epithelium of Norplant-treated subjects, where ET immunostaining was low while NEP immunoreactivity was increased. The latter may represent a local increase in enzyme activity, potentially explaining the reduced ET immunoreactivity. There was no correlation of ET immuno-reactivity with the duration of implant use or total number of bleeding days. The marked differences in the ET immunostaining pattern in Norplant users, with their increased risk of abnormal uterine bleeding, suggest that ET may be important in controlling menstrual bleeding. Whether endometrial epithelial cell ET has a role as a mitogen in endometrial repair and regeneration, or as a vasoconstrictor important in the cessation of bleeding following menstruation, remains to be determined.

Functional role of angiotensin ii type 1 and 2 receptors in the regulation of uterine blood flow in nonpregnant sheep

Functional role of angiotensin ii type 1 and 2 receptors in the regulation of uterine blood flow in nonpregnant sheep