Estradiol enhances T-type calcium channel activation in human myometrium telocytes.

Uterine peristalsis is essential for gamete transport and embryo implantation. It shares the characteristics of spontaneity, rhythmicity, and directivity with gastrointestinal peristalsis. Telocytes, the "interstitial Cajal-like cells" outside the digestive canal, are also located in the uterus and may act as pacemakers. To investigate the possible origin and regulatory mechanism of periodic uterine peristalsis in the human menstrual cycle, telocytes in the myometrium were studied to determine the effect of estradiol on T-type calcium channel regulation. In this study, biopsies of the human myometrium were obtained for cell culture, and double-labeling immunofluorescence screening was used to identify telocytes and T-type calcium channel expression. Intracellular calcium signal measurements and patch-clamp recordings were used to investigate the role of T-type calcium channels in regulating calcium currents with or without estradiol. Our study demonstrates that telocytes exist in the human uterus and express T-type calcium channels. The intracellular Ca2+ fluorescence intensity marked by Fluo-4AM was dramatically decreased by NNC 55-0396, a highly selective T-type calcium channel blocker, but enhanced by estradiol. T-type calcium current amplitude increased in telocytes incubated with estradiol in a dose-dependent manner compared to the control group. In conclusion, our study demonstrated that telocytes exist in the human myometrium, expressing T-type calcium channels and estradiol-enhanced T-type calcium currents, which may be a reasonable explanation for the origin of uterine peristalsis. The role of telocytes in the human uterus as pacemakers and message transfer stations in uterine peristalsis may be worth further investigation.

Human Myometrial Contractility Assays.

Traditional contractility assays using an organ bath setup consist of several chambers (or baths) perfused with temperature-controlled, oxygenated physiological saline. Strips or rings of tissue (usually smooth or cardiac muscle) are mounted within the organ bath between a fixed hook and an isometric force transducer. The contraction force is recorded by the transducer and different parameters of contraction are analyzed. Different experimental protocols can be performed to investigate the effect of drugs and reagents on tissue contractility to investigate tissue physiology or determine the in vivo potential of novel pharmaceutical compounds. Here, the application of a modified organ bath to measure ex vivo contractions of small strips of human uterine smooth muscle (myometrium) is described, as well as protocols to study the effect of oxytocin and uterine relaxants on contraction.

Effects of corticotropin-releasing hormone on the expression of adenosine triphosphate-sensitive potassium channels (Kir6.1/SUR2B) in human term pregnant myometrium.

OBJECTIVE: Corticotropin-releasing hormone (CRH) is a crucial regulator of human pregnancy and parturition. Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels are important for regulating myometrial quiescence during pregnancy. We investigated regulatory effects of different concentrations of CRH on KATP channel expression in human myometrial smooth muscle cells (HSMCs) in in vitro conditions. METHODS: After treating HSMCs with different concentrations of CRH (1, 10, 102, 103, 104 pmol/L), mRNA and protein expression of KATP channel subunits (Kir6.1 and SUR2B) was analyzed by reverse transcription-polymerase chain reaction and western blot. We investigated which CRH receptor was involved in the reaction and measured the effects of CRH on intracellular Ca2+ concentration when oxytocin was administered in HSMCs using Fluo-8 AM ester. RESULTS: When HSMCs were treated with low (1 pmol/L) and high (103, 104 pmol/L) CRH concentrations, KATP channel expression significantly increased and decreased, respectively. SUR2B mRNA expression at low and high CRH concentrations was significantly antagonized by antalarmin (CRH receptor-1 antagonist) and astressin 2b (CRH receptor-2 antagonist), respectively; however, Kir6.1 mRNA expression was not affected. After oxytocin treatment, the intracellular Ca2+ concentration in CRH-treated HSMCs was significantly lowered in low concentration of CRH (1 pmol/L), but not in high concentration of CRH (103 pmol/L), compared to control. CONCLUSION: Our data demonstrated the regulatory effect was different when HSMCs were treated with low (early pregnancy-like) and high (labor-like) CRH concentrations and the KATP channel expression showed significant increase and decrease. This could cause inhibition and activation, respectively, of uterine muscle contraction, demonstrating opposite dual actions of CRH.

Effects of corticotropin-releasing hormone on the expression of adenosine triphosphate-sensitive potassium channels (Kir6.1/SUR2B) in human term pregnant myometrium

OBJECTIVE: Corticotropin-releasing hormone (CRH) is a crucial regulator of human pregnancy and parturition. Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels are important for regulating myometrial quiescence during pregnancy. We investigated regulatory effects of different concentrations of CRH on KATP channel expression in human myometrial smooth muscle cells (HSMCs) in in vitro conditions. METHODS: After treating HSMCs with different concentrations of CRH (1, 10, 102, 103, 104 pmol/L), mRNA and protein expression of KATP channel subunits (Kir6.1 and SUR2B) was analyzed by reverse transcription-polymerase chain reaction and western blot. We investigated which CRH receptor was involved in the reaction and measured the effects of CRH on intracellular Ca2+ concentration when oxytocin was administered in HSMCs using Fluo-8 AM ester. RESULTS: When HSMCs were treated with low (1 pmol/L) and high (103, 104 pmol/L) CRH concentrations, KATP channel expression significantly increased and decreased, respectively. SUR2B mRNA expression at low and high CRH concentrations was significantly antagonized by antalarmin (CRH receptor-1 antagonist) and astressin 2b (CRH receptor-2 antagonist), respectively; however, Kir6.1 mRNA expression was not affected. After oxytocin treatment, the intracellular Ca2+ concentration in CRH-treated HSMCs was significantly lowered in low concentration of CRH (1 pmol/L), but not in high concentration of CRH (103 pmol/L), compared to control. CONCLUSION: Our data demonstrated the regulatory effect was different when HSMCs were treated with low (early pregnancy-like) and high (labor-like) CRH concentrations and the KATP channel expression showed significant increase and decrease. This could cause inhibition and activation, respectively, of uterine muscle contraction, demonstrating opposite dual actions of CRH.

A comparison of the effects of lindane and FeCl3/ADP on spontaneous contractions in isolated rat or human term myometrium.

Oxidative stress affects the contractile behavior of smooth muscle resulting in complications during labor. Toxicants such as lindane and ferric chloride (FeCl3)/adenosine diphosphate (ADP) cause oxidative stress and have previously been shown to inhibit smooth muscle contraction. In this study we examined the effects of the oxygen species scavengers, ascorbic acid and N-acetylcysteine on lindane and FeCl3/ADP's inhibition of spontaneous myometrial contractions in rat and human myometrium. Lindane and FeCl3/ADP gave rise to concentration-dependent reductions in rat (EC50 11.8×10-6M and 0.9×10-3M) and human myometrial contractions (EC50 16.3×10-6M and 1.1×10-3M, respectively). Pre-treatment with N-acetylcysteine significantly increased the EC50 for the effects of lindane on motility index of human tissue and reduced the maximum inhibitory effect of FeCl3/ADP on contractions in both rat and human myometrium. Ascorbic acid reduced the effects of FeCl3/ADP in rat tissue only. In conclusion pre-treatment with specific antioxidants may protect both rat and human myometrium from the inhibitory effects of lindane and FeCl3/ADP.

Lignocaine augments the in-vitro uterine contractions involving NO-guanylyl cyclase dependent mechanisms.

AIMS: Lignocaine is used during intrapartum and postpartum period but there are conflicting reports regarding the effect of lignocaine on uterine contractility. Therefore, this study was undertaken to delineate the effect of lignocaine on uterine contractility and the underlying mechanisms. MAIN METHODS: The in vitro contractions were recorded from the uterine segments obtained from adult rats (in estrous phase) and also from human myometrial tissue. Effect of lignocaine on spontaneous uterine contractions was recorded in the absence or presence of antagonists. Effect of sodium nitroprusside (SNP, NO donor) on uterine contractility was assessed. The NO2- was assayed (indicator of NO activity) from the supernatant after exposing the myometrial tissue to lignocaine in the absence or the presence of L-NAME or hemoglobin. KEY FINDINGS: Lignocaine (100µM) increased the amplitude of uterine contractions by 75% with no alterations in frequency. Similar magnitude of increase was seen with human myometrial tissue also. The spontaneous activities were absent in Ca2+-free or in nifedipine (10µM) containing medium. Heparin (IP3 blocker, 10IU/ml), but not the indomethacin (10µM) blocked the lignocaine-induced augmentation. L-NAME (NOS inhibitor, 10µM) or methylene blue (guanylyl cyclase inhibitor, 100µM) partially blocked the lignocaine-induced augmentation. SNP (30µM) increased the amplitude of spontaneous uterine contractions. Lignocaine increased the NO2- content (indicator of NO activity) of uterine tissue and the increase was blocked by L-NAME or hemoglobin. SIGNIFICANCE: Present observations indicate that lignocaine augments the amplitude of uterine contractions via Ca2+-dependent mechanisms involving NO-G cyclase-dependent mechanisms.

Effects of the KIR7.1 Blocker VU590 on Spontaneous and Agonist-Induced Contractions of Human Pregnant Myometrium.

KIR7.1, an inwardly rectifying K+ channel, plays a critical role in regulating uterine excitability during pregnancy and has been suggested as a potential new target for the treatment of conditions arising from dysfunctional uterine contractility, for example, atonic postpartum hemorrhage. The aim of this study was to investigate the effects of the selective KIR7.1 blocker, VU590, on both spontaneous and agonist-stimulated contractions of human pregnant myometrium in vitro. At a concentration of 20 µmol/L, VU590 significantly increased the mean contractile force and the frequency of spontaneous contractions ( P < 0.05) when compared to vehicle-treated tissues. However, there was a significant ( P < 0.0001) monoexponential decay in amplitude with time of exposure. When VU590 was coadministered with EC50 concentration of the uterotonics oxytocin, ergometrine, or carboprost, the only significant changes were an immediate decrease in the amplitude of oxytocin- and carboprost-induced contractions and a delayed reduction in amplitude and an increase in the frequency of ergometrine-induced contractions. Amplitude to all 3 agents in the presence of VU590 showed a monoexponential decay with time of exposure ( P < 0.0001). We conclude that VU590 modifies the contractility of pregnant human myometrium in support of a role for KIR7.1 in regulating that process. However, VU590 in vitro does not produce the types of contraction, either alone or in combination with other uterine stimulants that would suggest its usefulness as a first- or second-line clinical uterotonic agent.

The Synergic In Vitro Tocolytic Effect of Nifedipine Plus Ritodrine on Human Myometrial Contractility.

Many pharmacological agents have been investigated to manage preterm labor; we postulate that a combination of tocolytic drugs may achieve a better effect in the prevention of uterine contractions without dose-dependent adverse effects. The aim of this study was to evaluate the inhibitory effect of dual combinations of tocolytics in vitro. Human myometrium was obtained during elective cesarean sections (term without labor; n = 40). Myometrial strips were placed in organ baths for the measurement of isometric tension. Contractile activity was induced by oxytocin (10-8 mol/L), then a concentration-response curve to single or dual combinations of tocolytics was started. All studied tocolytics (nifedipine, ritodrine, nitroglycerin, atosiban, and NS-1619), when used alone, significantly inhibited myometrial contractions. When combined, nifedipine plus ritodrine produced a significantly greater inhibition of contractility than each drug alone in the midrange of concentrations. The combination of nifedipine plus nitroglycerin or nifedipine plus atosiban produced a significantly greater inhibition than nitroglycerin or atosiban alone but not greater than nifedipine. The combination of nifedipine plus NS-1619 (Ca+2-activated K+ [BKCa] channel opener) reduced the inhibitory effect of each drug. We concluded that a selected combination of tocolytics (nifedipine plus ritodrine) produced a significantly greater inhibitory effect on contractility than each drug alone at intermediate concentrations. Thus, specific combinations of tocolytics with different intracellular signaling pathways may have a synergic effect constituting a provocative new option for preterm labor treatment.

Inhibition of Uterine Contractility by Thalidomide Analogs via Phosphodiesterase-4 Inhibition and Calcium Entry Blockade.

Uterine relaxation is crucial during preterm labor. Phosphodiesterase-4 (PDE-4) inhibitors have been proposed as tocolytics. Some thalidomide analogs are PDE-4 inhibitors. The aim of this study was to assess the uterus-relaxant properties of two thalidomide analogs, methyl 3-(4-nitrophthalimido)-3-(3,4-dimethoxyphenyl)-propanoate (4NO2PDPMe) and methyl 3-(4-aminophthalimido)-3-(3,4-dimethoxyphenyl)-propanoate (4APDPMe) and were compared to rolipram in functional studies of spontaneous phasic, K⁺-induced tonic, and Ca2+-induced contractions in isolated pregnant human myometrial tissues. The accumulation of cAMP was quantified in HeLa cells. The presence of PDE-4B2 and phosphorylated myosin light-chain (pMLC), in addition to the effect of thalidomide analogs on oxytocin-induced pMLC, were assessed in human uterine myometrial cells (UtSMCs). Thalidomide analogs had concentration-dependent inhibitory effects on spontaneous and tonic contractions and inhibited Ca2+-induced responses. Tonic contraction was equipotently inhibited by 4APDPMe and rolipram (IC50 = 125 ± 13.72 and 98.45 ± 8.86 µM, respectively). Rolipram and the thalidomide analogs inhibited spontaneous and tonic contractions equieffectively. Both analogs increased cAMP accumulation in a concentration-dependent manner (p < 0.05) and induced changes in the subcellular localization of oxytocin-induced pMLC in UtSMCs. The inhibitory effects of thalidomide analogs on the contractions of pregnant human myometrium tissue may be due to their PDE-4 inhibitory effect and novel mechanism as calcium-channel blockers.

In vitro contractile effects of agents used in the clinical management of postpartum haemorrhage.

Uterine atony is a major cause of postpartum haemorrhage and maternal mortality. However, the comparative pharmacology of agents used to treat this condition is poorly understood. This study evaluates, using human pregnant myometrium in vitro, a range of contractile parameters for agents used in the clinical treatment of atonic postpartum haemorrhage. The effects of oxytocin, carbetocin, ergometrine, carboprost, syntometrine and misoprostol were investigated in 146 myometrial strips from 19 donors. The potency and maximal response values were obtained, and compared, using both maximal amplitude and mean contractile force as indices of contraction. Single, EC50 concentrations of the agents were administered and both force and contraction peak parameters were compared during a 15-min exposure. Differences were considered significant when P<0.05. There were no significant differences in the peak amplitude of response between agents, except for misoprostol, which was inactive. There was a wide difference in potencies using both measures of contractility, with oxytocin and carbetocin being the most potent. The most important difference between the agents was in their ability to increase the mean contractile force, with oxytocin superior to all agents except syntometrine. In single dose experiments, mean contractile force was the parameter that separated the agents. In this respect, oxytocin was not statistically different from carboprost or syntometrine, but was superior to all other agents. These findings support a clear role for oxytocin as the first line agent for treatment of postpartum haemorrhage and raise doubts about the potential clinical usefulness of misoprostol.

Steroid receptor co-activator interacting protein (SIP) mediates EGF-stimulated expression of the prostaglandin synthase COX2 and prostaglandin release in human myometrium.

STUDY HYPOTHESIS: Steroid receptor coactivator interacting protein (SIP/KANK2) is involved in regulating the expression of the prostaglandin (PG)-endoperoxide synthase 2 (PTGS2; also known as cyclo-oxygenase 2, COX2) and PG release in human myometrium. STUDY FINDING: SIP is phosphorylated in myometrial cells in response to epidermal growth factor (EGF)-stimulation and is required for EGF-stimulated increases in COX2 expression, PGE2 and PGF2α release, and expression of interleukins (IL) 6 and IL8. WHAT IS KNOWN ALREADY: Human parturition involves inflammatory and non-inflammatory pathways and requires activation of the intrauterine PG cascade. A key mediator of uterine PG production is the highly inducible enzyme COX2. Regulation of COX2 expression is complex, and novel factors involved in its induction may play an important role during labour. The expression and function of SIP in uterine tissues has never been investigated. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Mass spectrometry was used to identify SIP from cultured primary myometrial cells, and its expression in fresh placenta, fetal membranes, decidua and myometrium from pregnant and non-pregnant women was determined by western blotting. SIP expression in myometrial cells was reduced using small interfering RNA (siRNA), and COX2 expression was stimulated with EGF. COX2, IL6 and IL8 mRNA and COX2 protein expression were measured using quantitative RT-PCR (RT-qPCR) and western blotting respectively, and release of PGE2 and PGF2α by enzyme immunoassay. The time course and dose response of SIP phosphorylation in response to EGF were determined, and phosphorylation was measured in the presence of the mitogen-activated protein kinase kinase 1(MEK1) inhibitor PD-184352. Fresh myometrial tissue was used to confirm effects of EGF and MEK1 inhibition on SIP phosphorylation and COX2 expression. A profile of transcription factor (TF) activity after SIP knockdown was carried out using a commercially available array. MAIN RESULTS AND THE ROLE OF CHANCE: We have demonstrated expression of SIP in human myometrium. siRNA-mediated knockdown of SIP resulted in decreased EGF-stimulated COX2 protein expression (P < 0.001), and decreased release of PGE2 (P < 0.001) and PGF2α (P < 0.01). EGF stimulation resulted in rapid and transient phosphorylation of SIP, which was blocked by pharmacological inhibition of the MEK1/ERK (extracellular signal-regulated kinase) signalling pathway with PD-184352 (P < 0.001). Moreover inhibition of ERK signalling significantly decreased EGF-stimulated COX2 expression (P < 0.001). EGF phosphorylated SIP and increased COX2 expression in a MEK1/ERK-dependent manner in freshly isolated pregnant myometrium. Our data have uncovered a pathway mediating EGF-stimulated COX2 expression that is ERK and SIP dependent, providing a novel function for SIP in the pregnant uterus. Furthermore, EGF stimulated the expression of IL6 and IL8 mRNA in a SIP-dependent manner (both P < 0.05), and SIP expression was positively associated with activation of serum response factor (SRF) and YY1 TF (P < 0.001 and P < 0.05, respectively), suggesting additional important roles for myometrial SIP. LIMITATIONS, REASONS FOR CAUTION: While we describe a new role for myometrial SIP, we are yet to determine whether SIP phosphorylation is required for its effects on regulating COX2 expression and PG release. Our data are from in vitro studies using fresh tissue and cultured myometrial cells therefore may not fully reflect the conditions in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Our group has previously described increases in myometrial COX2 expression with labour at term and preterm. EGF levels rise in the amniotic fluid near term suggesting it may participate in paracrine signalling events, altering gene expression in the myometrium. Our novel data describe a role for SIP in regulating EGF-stimulated expression of myometrial COX2 and PG release. Moreover, our profile of SIP-dependent TF activation provides a platform for further investigations into additional roles for SIP in uterine function. These findings may facilitate the development of new, targeted drugs for the management of labour. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: This work was supported by an Action Medical Research grant (SP4612). The authors have no competing interests to declare.

Why are We Waiting to Start Large Scale Clinical Testing of Human Chorionic Gonadotropin for the Treatment of Preterm Births?

Preterm births are an expensive global health problem. Despite the basic science and clinical research advances to better understand and prevent preterm births, the rates are increasing. There are several therapeutic options. While some options such as progestins work for selected women, others such as magnesium sulfate can only be used for delaying births for 24 to 48 hours so that the patients can be treated with corticosteroids to promote fetal lung maturity. Based on the scientific and clinical evidence, we recommend testing human chorionic gonadotropin in a large multicenter, randomized, double-blind, and placebo-controlled clinical trials in women with active preterm labor and those with a previous history of preterm births. Human chorionic gonadotropin is not only inexpensive but also has not shown any side effects so far in the infants or in the mothers.

Differing In Vitro Potencies of Tocolytics and Progesterone in Myometrium From Singleton and Twin Pregnancies.

We compared the relaxant effect of 2 known tocolytics; indomethacin and atosiban and progesterone, on pregnant human myometrial spontaneous and oxytocin-induced contractions from singleton and twin pregnancies. All agents exerted a concentration-dependent relaxant effect on myometrial contractions. There was no significant difference in the concentration-response curves between singletons and twins for progesterone or indomethacin on spontaneous contractions or atosiban on oxytocin-induced contraction. Under oxytocin however, the concentration-response curves for indomethacin and progesterone were significantly shifted to the right for both amplitude of contraction (P < .01) and activity integral (P < .01). When compared to singleton myometrium however, the concentration-response curves were significantly shifted to the right in the twin myometrium group (P < .05 progesterone and P < .001 indomethacin). We conclude that a greater concentration of progesterone and indomethacin is required to inhibit oxytocin-induced myometrial contractions in twins compared to singletons in vitro. The differences noted in the tissue pharmacologies may have implications for the successful prevention or inhibition of preterm labor in twin pregnancy.

Stro-1/CD44 as putative human myometrial and fibroid stem cell markers.

OBJECTIVE: To identify and characterize myometrial/fibroid stem cells by specific stem cell markers in human myometrium, and to better understand the stem cell contribution in the development of uterine fibroids. DESIGN: Prospective, experimental human and animal study. SETTING: University research laboratory. PATIENT(S)/ANIMAL(S): Women undergoing hysterectomy for treatment of symptomatic uterine fibroids and female NOD/SCID/IL-2Rγ(null) mice. INTERVENTION(S): Identification and isolation of stem cells from human fibroids and adjacent myometrium tissues using Stro-1/CD44-specific surface markers. MAIN OUTCOME MEASURE(S): Flow cytometry, semiquantitative polymerase chain reaction, clonogenicity assays, cell culture, molecular analysis, immunocyto-histochemistry, in vitro differentiation, and xenotransplantation assays. RESULT(S): Using Stro-1/CD44 surface markers, we were able to isolate stem cells from adjacent myometrium and human fibroid tissues. The undifferentiated status of isolated cells was confirmed by the expression of ABCG2 transporter, as well as additional stem cell markers OCT4, NANOG, and GDB3, and the low expression of steroid receptors ERα and PR-A/PR-B. Mesodermal cell origin was established by the presence of typical mesenchymal markers (CD90, CD105, and CD73) and absence of hematopoietic stem cell markers (CD34, CD45), and confirmed by the ability of these cells to differentiate in vitro into adipocytes, osteocytes, and chondrocytes. Finally, their functional capability to form fibroid-like lesions was established in a xenotransplantation mouse model. The injected cells labeled with superparamagnetic iron oxide were tracked by both magnetic resonance imaging and fluorescence imaging, thus demonstrating the regenerative potential of putative fibroid stem cells in vivo. CONCLUSION(S): We have demonstrated that Stro-1/CD44 can be used as specific surface markers to enrich a subpopulation of myometrial/fibroids cells, exhibiting key features of stem/progenitor cells. These findings offer a useful tool to better understand the initiation of uterine fibroids, and may lead to the establishment of effective therapeutic options.

Maternal Age and Contractility of Human Myometrium in Pregnancy.

There is controversy as to whether maternal age exerts an influence on the contractility of human myometrium in pregnancy. The aim of this study was to examine a series of functional contractile parameters of human myometrium in vitro, over a broad range of maternal ages. Myometrial tissue specimens were obtained at cesarean delivery from 32 women with maternal ages ranging from 28 to 52 years. Using in vitro recordings, a number of contractile parameters including maximal amplitude, mean contractile force, time to maximal amplitude, maximum rate of rise, and occurrence of simple and complex (biphasic and multiphasic) contractions were examined for spontaneous and induced contractile activity. The relationship between maternal age and individual parameters was evaluated using linear regression analysis. For all contractile parameters examined, for both spontaneous and induced contractions, no significant correlation was observed with maternal age between 28 and 52 years. The mean maximum amplitude values for spontaneous and oxytocin-induced contractions were 23 ± 3 and 43 ± 5 mN, respectively. The mean contractile forces for spontaneous and oxytocin-induced contractions were 1.5 ± 0.2 and 6.5 ± 0.9 mN, respectively. There was no variation in the proportion of biphasic or multiphasic contractions with maternal age. These results indicate there is no significant functional impairment of uterine contractility and no lack in responsiveness of myometrium in vitro, in the older mother. These findings do not support the concept that there may be a biological basis for dysfunctional labor or increased cesarean delivery rates in older parturients.

Cathepsin B in human myometrium and in uterine leiomyomas at various stages of tumour growth.

OBJECTIVE: Cathepsin B is a major cysteine protease involved in the degradation of extracellular matrix proteins, as well as in the activation of precursor forms of other proteases and in release of matrix-bound growth factors. We assessed the expression and activity of cathepsin B, and the inhibitory effect of cysteine protease inhibitors in human myometrium and uterine leiomyomas at various stages of tumour growth. STUDY DESIGN: Studies were performed on human myometrium collected from 12 patients and on uterine leiomyomas of various weights: small (less than or equal to 25 g, taken from 10 patients) and large (more than or equal to 100 g, obtained from 13 patients). Tissue extracts were assayed for cathepsin B activity and for inhibitory effect of cysteine protease inhibitors against papain using fluorogenic substrates, and calculated per DNA content. Statistical analysis was performed by Kruskal-Wallis analysis of variance followed by Dunn's post hoc tests. The enzyme expression was evaluated by SDS/polyacrylamide gel electrophoresis followed by Western immunoblotting. RESULTS: In all the investigated tissues cathepsin B exists mainly in a fully processed double-chain form. The enzyme activity and expression were similar in control myometrium and in small leiomyomas. However, they distinctly increased during tumour growth. The effect of cysteine protease inhibitors was comparable in all the tissues examined. CONCLUSION: These data suggest that the enhanced activity and expression of cathepsin B but not the action of cysteine protease inhibitors contribute to an increased remodelling of extracellular matrix and bioavailability of various growth factors, which favour leiomyoma growth.

Overexpression of the truncated form of High Mobility Group A proteins (HMGA2) in human myometrial cells induces leiomyoma-like tissue formation.

The pathogenesis of uterine leiomyomas, the most common benign tumor in women, is still unknown. This lack of basic knowledge limits the development of novel non-invasive therapies. Our group has previously demonstrated that leiomyoma side population (SP) cells are present in tumor lesions and act like putative tumor-initiating stem cells in human leiomyoma. Moreover, accumulated evidence demonstrates that these benign tumors of mesenchymal origin are characterized by rearrangements of the High Mobility Group A proteins (HMGA). In this work, we tested the hypothesis that leiomyoma development may be due to overexpression of HMGA2 (encoding high mobility group AT-hook2) in myometrial stem cells using in vitro and in vivo approaches. Our work demonstrates that the truncated/short form of HMGA2 induces myometrial cell transformation toward putative tumor-initiating leiomyoma cells and opens up new possibilities to understand the origin of leiomyomas and the development of new therapeutic approaches.

The effects of methyl palmitate, a putative regulator from perivascular fat, on the contractility of pregnant human myometrium.

AIMS: Methyl palmitate is thought to cause relaxation in vascular smooth muscle by opening voltage-activated potassium channels. We have tested the hypothesis that methyl palmitate, a putative regulator from perivascular fat, is an inhibitor of the contractility of human pregnant myometrium and that its effects might partially explain the higher incidence of dysfunctional labor in obese women compared to those with normal body mass indices. MAIN METHODS: Strips of myometrium obtained with informed consent from women undergoing elective cesarean section at term were mounted in organ baths. Strips stimulated with oxytocin (1nM) or KCl (30mM) were exposed to cumulatively increasing concentrations of methyl palmitate up to 10µM. Similar strips were exposed to cumulative addition of the potassium channel blockers 4-aminopyridine and tetraethylammonium. The contractility of the strips was monitored and analyzed using conventional methods. KEY FINDINGS: Methyl palmitate failed to inhibit oxytocin- or KCl-induced contractions over the concentration range tested. In fact, it exerted a slight excitatory effect in the presence of KCl, though not in the presence of oxytocin. The contractility of naïve strips was unaltered by exposure to 1µM methyl palmitate. Both 4-aminopyridine and tetraethylammonium produced concentration-dependent contractions of human pregnant myometrium providing pharmacological evidence for the presence of voltage-activated potassium channels in this preparation. SIGNIFICANCE: Our findings do not support the hypothesis that methyl palmitate is an inhibitor of human pregnant myometrial contractility. Alternate hypotheses must be pursued to explain the higher incidence of dysfunctional labor in obese women.

The effects of extracellular calcium-sensing receptor ligands on the contractility of pregnant human myometrium in vitro.

Ligands for extracellular calcium-sensing (CaS) receptors inhibit oxytocin-induced contractions of the rat's uterus. In this study, we investigated whether the CaS receptor ligands calindol, cinacalcet, and calhex 231 have similar effects on pregnant human myometrium. We compared their effects to those of the calcium-channel blocker nifedipine. In conventional concentration-effect experiments, both the mean contractile force (MCF) and the maximum amplitude of contractions induced by 1 nmol/L oxytocin were inhibited by nifedipine. Calindol and cinacalcet were ineffective as inhibitors, while calhex-231 produced partial inhibition. When single 10 µmol/L doses were applied calhex-231 produced a slowly developing inhibition, reducing the MCF to 38%, and amplitude to 34%, of vehicle controls after 1 hour. In similar experiments, calindol was ineffective while cinacalcet weakly inhibited only the amplitude. Immunohistochemistry revealed sparse expression of CaS receptors in pregnant human myometrium.

Spontaneous and induced labour are associated with different myometrial proteomes in the human.

Human myometrium undergoes a major phenotypic change at labour likely involving modifications to key regulatory proteins. In some cases, the myometrium fails to activate normally and medical intervention is required to induce labour. In this study, 2-D DIGE was used to examine changes in the myometrial proteome at the time of spontaneous (SL) and induced labour (IL). Proteomic profiles of nonlabouring term myometria (NL, n = 6) were quantitatively compared to SL (n = 6) and prostaglandin/oxytocin-IL term myometria (n = 6). In SL samples, 23 differentially expressed protein spots were detected (9 increased/14 decreased compared to NL, p<0.05). In IL samples, 59 differentially expressed spots were observed (13 increased/46 decreased compared to NL). Comparison of SL and IL proteomes revealed 69 differentially expressed proteins (7 increased/62 decreased). Two proteins consistently decreased in SL and IL samples were identified as transgelin (1.98- and 1.97-fold decrease in SL and IL, respectively) and αB-crystallin (3.27- and 2.49-fold decrease). Levels of desmin and cytosolic phospholipase A2 ß were decreased 2.9- and 2.65-fold, respectively only in IL samples. Our results show human labour is accompanied by general downregulation of specific myometrial proteins. Differences exist between SL and IL myometrial proteomes indicating divergence of underlying processes and highlighting the importance of distinguishing these groups in future studies of parturition. Our findings underscore the utility of discovery approaches in investigations of organ-wide protein changes that underlie discrete physiological events including human labour.