Endothelin synthesis and receptors in human endometrium throughout the normal menstrual cycle

This study was undertaken to investigate the presence of messengerRNA (mRNA) for prepro-endothelin-l (ET-1) and the known receptorsubtypes (ET_A and ET_B) in human endometrium at different stagesof the menstrual cycle obtained at hysterectomy. Northern blotanalysis revealed expression of ET-1 mRNA in human endometriumduring the normal menstrual cycle. The concentration of ET-1mRNA in endometrial tissue was greater during the menstrualand proliferative phases than during the ovulatory and secretoryphases. Immunoreactive ET-1 was secreted into the medium ofisolated endometrial stromal cells. Oestradiol and progesteronesignificantly attenuated ET-1 release in endometrial stromalcells cultured for 6 days. ET_A and ET_B mRNA were also presentin endometrial tissue of the normal cycle. The concentrationof ET_A receptor mRNA was greater in the proliferative phasethan in the secretory phase, whereas expression of ET_B mRNAincreased in menstrual phase. ET-1 significantly increased extracellularaccumulation of cyclic AMP (cAMP), intracellular generationof inositol phosphates and significantly enhanced DNA synthesisin cultured endometrial stromal cells from the proliferativephase. Our results showed that human endometrial cells synthesizedand released ET-1, and contained ET_A and ET_B receptors whichwere functionally coupled to phosphoinosttide breakdown andto adenylate cyclase with the increase of cAMP by ET-1 stimulation.Our findings suggest that ET-1 may have a potential autocrineand/or paracrine function in human endometrial stromal cells. cyclic AMP/endothelin-l synthesis/human endometrium/inositol phosphate/receptors     

Endocrinology and paracrinology: Localization of endothelin receptors in human uterus throughout the menstrual cycle

Quantitative autoradiography employing the ET_A selective ligand[¹²⁵I]PD151242 and the ET_B selective ligand [¹²⁵I]BQ3020 wasused to assess the localization of ET_A and ET_B receptors inhuman uterus throughout the menstrual cycle. ET_A and ET_B receptorswere present in endometrium and myometrium across the menstrualcycle. In myometrium, neither ET_A nor ET_B receptor density showedany detectable change across the menstrual cycle. ET_A receptorswere expressed in stroma throughout the endometrium and showedan increase in density in proliferative endometrium comparedwith secretory and menstrual endometrium. Endometrial ET_B receptorswere expressed at low density in the proliferative phase. Inthe early secretory phase there was an increase in ET_B receptordensity in the glandular epithelium of the basal region of theendometrium but not in functional endometrium. In the late secretoryphase ET_B receptor expression was increased in glandular epitheliumthroughout the endometrium. The highest density of ET_B receptorswas seen in menstrual endometrium, where they were present instromal as well as epithelial cells. These results suggest thatovarian steroid hormones may play a role in the control of expressionof ET_A and ET_B receptors in endometrial stromal and epithelialcells respectively. endothelium/endothelium receptors/human/uterus     

Secretory endometrium highly expresses urocortin messenger RNA and peptide: possible role in the decidualization process

BACKGROUND: Urocortin (UCN) gene expression and synthesis have been reported in epithelial and stromal cells of the human endometrium. In this study we evaluated (i) UCN messenger RNA (mRNA) expression and peptide production in uterine specimens collected throughout the endometrial cycle, (ii) UCN secretion after decidualization of cultured human endometrial stromal cells (HESCs) and (iii) the effect of UCN on endometrial decidualization. METHODS: HESCs were isolated from samples of human endometrium collected from healthy patients with normal menstrual cycle and cultured in presence of cAMP, 17-beta-estradiol (E(2)) + medroxyprogesterone acetate (MPA) and UCN. UCN levels were measured in endometrial extracts by an enzyme immunoassay, and changes of endometrial UCN mRNA expression were measured by RT-PCR analysis. RESULTS: UCN peptide concentrations and mRNA expression were highest in the secretory phase of the menstrual cycle (P < 0.001, late secretory versus early and late proliferative phase) and higher in the late than the early secretory phase (P < 0.01). After decidualization of HESC with cAMP or E(2) + MPA, UCN levels rose in parallel with prolactin concentrations by days 6 (P < 0.01, for all). Finally, the addition of UCN to HESCs, with or without E(2) + MPA, induced the release of prolactin. CONCLUSIONS: The evidence that (i) UCN is highly expressed in the secretory phase of the endometrial cycle; (ii) cAMP and E(2) + MPA modulate secretion of UCN and (iii) UCN induces HESCs decidualization together suggest a possible role for UCN in endometrial physiology.     

Human endometrial stromal cells as a source of soluble intercellular adhesion molecule (ICAM)-l molecules

Intercellular adhesion molecule (ICAM)-l-mediated cell-celladhesion is essential for various immunological functions, includingnatural killer (NK) cell-mediated cyto-toxicity against endometrium.The present study was designed to establish whether sheddingof ICAM-1 from cultured endometrial stromal cells occurred andto characterize its potential functional significance in endometrialphysiology and pathology. The shed sICAM-1 molecule was detectedand quantified in supernatants from endometrial stromal culturesand in peritoneal fluids by a specific enzyme-linked immunosorbentassay. The results of this study indicate that cultured endometrialstromal cells constitutively shed ICAM-1 from their surface.This ability is regulated during the menstrual cycle, as itappears to be higher in the proliferative than in the secretoryphase of the cycle (16.93 ± 2.2 and 7.7 ± 1.76ng/ml respectively). In order to evaluate whether the releaseof sICAM-1 could interfere with cell-mediated lysis of endometrium,we compared the determinations of sICAM-1 in endometrial supernatantswith the ability of such supernatants to suppress NK cell-mediatedcytotoxidty toward endometrial targets. A significant correlation(r = 0.6, P < 0.05) was found between the sICAM-1 concentrationin endometrial supernatants and the percentage of inhibitionof NK cell-mediated lysis exerted by the same supernatant samples.Finally, endometrial stromal shedding of sICAM-1 appears tobe related to endometriosis since endometrial stromal culturesobtained from patients with advanced stages of the disease releasedsignificantly higher amounts of the soluble protein comparedto the control group (P < 0.05). sICAM-1 is a soluble moleculewhich can interfere with immunological functions, and its sheddingmay be one of the mechanisms by which refluxed endometrial cellsescape immunosurveillance.     

Lack of correlation between vascular endothelial growth factor production and endothelial cell proliferation in the human endometrium.

The role of vascular endothelial growth factor (VEGF) in endometrial angiogenesis was examined by measuring its production in human endometrial tissues from different stages of the menstrual cycle and relating these data to endothelial cell proliferation in the same tissues. Conditioned medium was collected from explant, and separated glandular epithelial and stromal cells cultured from 24 normal human endometrial biopsies and VEGF measured by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was also used to assess VEGF and the percentage of proliferating microvessels in the samples. Wide variation in results between individual endometrial samples at each stage of the menstrual cycle was observed for all parameters measured. There was no significant difference in VEGF secretion by explant, glandular epithelial or stromal cell cultures across the menstrual cycle, or in the percentage of proliferating vessels. VEGF immunostaining in the stroma was elevated during the early proliferative stage (P = 0.03). Epithelial cells secreted more VEGF than stromal cells (1.76 +/- 0.46 versus 0.46 +/- 0.06 ng per 10(5) cells; P = 0.002). There was no correlation between VEGF secreted by cultured explants, epithelial or stromal cells, VEGF immunostaining and the proportion of proliferating microvessels. These results show that the majority of endometrial VEGF is produced by glands, but neither total glandular nor stromal VEGF is correlated with endometrial endothelial cell proliferation. There is still no clear understanding on the regulation of human endometrial angiogenesis.     

Tissue inhibitor of metalloproteinases (TIMP)-1, -2 and -3 in human endometrium during the menstrual cycle

The extensive remodelling of the human endometrium throughout the menstrual cycle is accompanied by changes in production of matrix metalloproteinases, the activity of which can be inhibited by specific tissue inhibitors or by tissue inhibitors of metalloproteinases (TIMP)s with a 1:1 stoichiometry. This study immunolocalized TIMP-1, TIMP-2 and TIMP-3 in dated normal human endometrium across the menstrual cycle and examined cultured endometrial cells for their production. All three TIMPs were present in the major cellular compartments, luminal epithelium, glands, stroma, endothelial cells and vascular smooth muscle cells with the most intense immunoreactivity in the luminal epithelium. TIMP-1 and -3 were lower in the mid-to-late proliferative phase with a nadir of TIMP-3 particularly in the late proliferative phase. Decidualized stromal cells stained strongly positive for TIMP-1, -2 and -3. Cells of haematopoietic origin never stained. Monensin treatment of tissue resulted in accumulation of TIMPs in all cellular compartments but particularly of TIMP-1 in epithelium. Cultured endometrial stromal cells released more TIMP-1 than TIMP-2 or TIMP-3 into culture medium and all were increased following decidualization in vitro. Epithelial cells in culture produced less TIMPs than stromal cells, and only a few epithelial cells in each culture were immunopositive for TIMP-1. The ubiquitous distribution of TIMPs implicates them in maintenance of endometrial integrity, with changes in the matrix metalloproteinases without concomitant changes in TIMPs determining endometrial matrix degradation.      

Expression and function of interleukin-11 and its receptor alpha in the human endometrium

The interleukin-11 (IL-11) receptor alpha has an important function in decidualization of mouse endometrial stroma but the function of IL-11 and its receptor in the human endometrium remains unknown. The mRNA for IL-11 and its receptor alpha in human endometrial tissue samples were analysed by semi-quantitative RT-PCR and RNase protection assays respectively. The proteins were detected in frozen endometrial tissue samples by immunofluorescence. The effect of heparin-binding epidermal growth factor (HB-EGF) on secretion of IL-11 by cultured endometrial stromal cells was assessed by enzyme-linked immunosorbent assay. The proliferative potential of IL-11 in endometrial stromal cells was assessed by [(3)H]thymidine uptake. IL-11 and its receptor alpha mRNAs and proteins were detected in the endometrium throughout the cycle. Distinct patterns of localization of the ligand and receptor were observed. HB-EGF induced IL-11 secretion by cultured stromal cells, and IL-11 induced [(3)H]thymidine uptake by these cells. Our data suggest that IL-11-receptor interactions may perform different functions in the human endometrium at different stages of the cycle, and that secretion of IL-11 is modulated by local growth factors.     

Endocrinology and pacrinology: Localization of bradykinin type II receptor mRNA in human endometrium

Bradykinin is a nonapeptide inflammatory agent that in the endometriumstimulates stromal cell proliferation, prostaglandin synthesisand electrogenic ion transport. The expression of bradykinintype II (B₂) receptor mRNA was examined by in-situ hybridizationusing ³⁵S-labelled riboprobe in human endometrium to determineits temporal and spatial pattern of distribution throughoutthe menstrual cycle. The B₂ receptor mRNA was expressed in proliferativeand secretory endometrium. In the early proliferative endometriumthere were low levels of B₂ receptor mRNA over both the glandsand stromal cells. The signal increased in intensity and waslocalized over the glands with low levels of hybridization inthe stroma in the late proliferative endometrium. In the earlysecretory endometrium B₂ receptor mRNA was highly expressedin the endometrial glands. The strong hybridization signal persistedand in late secretory endometrium both glandular and stromalcells expressed B₂ receptor mRNA. The apparent increase in B₂receptor mRNA in the secretory endometrium suggests that bradykininacting via B₂ receptor may play a role in the increased vascularpermeability and vasodilatation associated with implantation. bradykinin/endometrium/implantation/menstruation/myometrium     

Urocortin 2 and urocortin 3 in endometriosis: evidence for a possible role in inflammatory response

Urocortin 2 (Ucn 2) and urocortin 3 (Ucn 3) are neuropeptides expressed by human endometrium. This study evaluated (i) the expression of Ucn 2 and Ucn 3 mRNA in endometriotic lesions and in endometrium of women with endometriosis; (ii) the effect of Ucn 2 and Ucn 3 on cytokines secretion from cultured endometrial stromal cells. Endometriotic tissue was collected from endometrioma (n=39); endometrial specimens were obtained from women with (n=39) and without (n=41) endometriosis throughout menstrual cycle. Tissue specimens were analysed for Ucn 2 and Ucn 3 mRNA expression and peptide localization; the effects of Ucn 2 or Ucn 3 on tumour necrosis factor (TNF-α) and interleukin (IL-4) secretion from cultured endometrial stromal cells was studied. Ucn 2 and Ucn 3 mRNA expression and localization were assessed by RT-PCR and by immuohistochemistry, respectively; cytokines secretion were measured by ELISA. Results showed that endometriotic tissue expressed both Ucn 2 and Ucn 3, with Ucn 3 expression higher in ectopic than in eutopic endometrium. Endometrial Ucn 2 mRNA expression in controls showed peak values at early proliferative phase, while in endometriotic patients low expression and no significant changes throughout menstrual cycle were observed. Endometrial Ucn 3 mRNA expression was highest in late secretory phase in controls, while in endometriotic patients low levels and no menstrual-cycle-related changes were found. When added to cultured endometrial cell cultures, Ucn 2 significantly increased TNF-α (P<0.01) and IL-4 (P<0.001), while Ucn 3 induced an increase of IL-4 secretion (P<0.01). In conclusion, endometriotic tissue expressed and localized Ucn 2 and Ucn 3; patients with endometriosis showed Ucn 2 and Ucn 3 mRNA expression in eutopic endometrium lower than in control group, with no endometrial cycle-related changes. Ucn 2 and Ucn 3-modulated TNF-α and IL-4 secretion from culture endometrial cells. These data suggest a possible involvement of Ucn 2 and Ucn 3 in the mechanisms of endometriosis.     

Cell cycle: Immunolocalization of bcl-2 protein in human endometrium in the menstrual cycle and simulated early pregnancy

Cell death by apoptosis is now regarded as an important featureof normal endometrial physiology. Recent reports have suggestedthat bcl-2, a proto-oncogene responsible for the suppressionof apoptosis, is expressed in endometrium and may be involvedin the regulation of menstruation. Using standard immunohistochemicalprocedures, the immunoreactivity of bcl-2 and progesterone receptorshas been investigated in normal human endometrium throughoutthe menstrual cycle (n = 25) as well as endometrium exposedto continued oestradiol and progesterone stimulation by ‘rescue’of corpus luteum (n = 4) with exogenous human chorionic gonadotrophin(HCG) administration (pseudopregnancy). Marked immunoreactivity,consistent with previous reports, was noted in the glandularepithelium during the proliferative phase of the cycle. Immunostainingpersisted in the glandular epithelium during the secretory phase,although the percentage and intensity of staining was markedlyreduced. Staining in the stromal compartment was only notedduring the late secretory phase of the cycle. Co-localizationwith an antibody against CD56 demonstrated that this immunoactivitylargely reflected the presence of lymphocytes in the stroma.Endometrium from subjects who underwent ‘luteal rescue’displayed limited immunostaining in either glands or stroma.The absence of significant bcl-2 expression in endocrinologicallymaintained endometrium makes it highly unlikely that bcl-2 isimportant in prolonging endometrial cell survival in the lutealphase of the menstrual cycle.     

Implantation: Stimulation of human endometrial epithelial cell interleukin 6 production by interleukin 1 and placental protein 14

The effect of interleukin 1 (ILI) and placental protein 14 (PP14)on the production of interleukin 6 (IL6) by cultured human endometrialepithelial cells prepared from endometrial biopsy material obtainedat different stages in the menstrual cycle was investigated.Basal IL6 production by cells prepared from proliferative endometriumwas greater than that produced by cells prepared from secretoryendometrium (7.3 ± 0.3 and 1.1 ± 0.2ng/well/24h respectively, P < 0.001). IL1 (0.025–2.5 ng/ml) causeda dose-dependent increase in IL6 production by cells preparedfrom both proliferative and secretory endometrium, but cellsprepared from secretory endometrium responded to a lower concentrationof ILI than those prepared from proliferative endometrium. ILI-stimulatedIL6 production by epithelial cells prepared from secretory endometriumtypically reached 10 times basal values, while in cells preparedfrom proliferative endometrium stimulated levels were approximatelytwice the basal values. PP14 (1–50 µg/ml) also causeda dose-dependent increase in IL6 production by epithelial cellsprepared from secretory endometrium, but had no effect on IL6production by cells prepared from proliferative endometrium.Even in secretory cells PP14 was less effective than IL1 atstimulating IL6 production, with stimulated levels only reachingtwice the basal values. This suggests that PP14 and IL1 actvia different mechanisms in the stimulation of IL6 production.The results show that IL6 production by human endometrial epithelialcells is stimulated by other immunomodulatory peptides and thismay be part of the network of such peptides in the endometriumwhich may influence embryo implantation.     

Molecular aspects of implantation: Variation in the expression of cellular retinoid binding proteins in human endometrium throughout the menstrual cycle

Human endometrium is a glandular epithelial tissue with a substantialunderlying stroma. Under the influence of ovarian steroids,endometrium undergoes a cyclical pattern of proliferation followedby secretory differentiation. Since retinoids promote the differentiationof many epithelia to secretory phenotypes they may be involvedin controlling the secretory differentiation of human endometrialepithelium. Cytosolic binding proteins for retinol (cellularretinol binding protein) and retinoic acid (cellular retinoicacid binding protein) may play an important part in regulatingthe availability of retinoic acid to its nuclear receptors andwe have therefore asked whether expression of mRNA for theseproteins varies in relation to endometrial differentiation.In a series of 54 endometrial biopsies, both endometrial epithelialand stromal cells expressed mRNA for cellular retinol bindingprotein type I at a constant level throughout the menstrualcycle. Cellular retinoic acid binding protein type II was alsoexpressed but the level of expression varied dramatically, beingelevated in the proliferative phase and depressed during thesecretory phase of the menstrual cycle in both epithelial andstromal cells. These data suggest that cytosolic binding proteinsmodulate the supply of retinoic acid to the nuclei of endometrialcells during the menstrual cycle and that retinoic acid is involvedin the cyclical control of endometrial differentiation.     

Uterus and endometrium: Inhibition of human endometrial stromal cell proliferation by interleukin 6

We investigated the ability of interleukin 6 (IL-6) to modulatehuman endometrial stromal cell growth in vitro Stromal cellproliferation in response to treatment with varying concentrationsof IL-6 was determined. Endometrial tissue was obtained from10 normally cycling women during the secretory phase of theirmenstrual cycle. Treatment with IL-6 resulted in a dose- andcell-density-dependent inhibition of endometrial stromal cellproliferation in vitro. The maximal inhibition was observedwith 200 pg/ml of IL-6 and at a concentration of 10⁵ cells/well.During in-vitro culture, stromal cells produced low amountsof IL-6 and demonstrated the presence of IL-6 receptor. Thesedata demonstrate that IL-6 acts as a growth-regulatory signalfor human endometrial stromal cells. We postulate that IL-6may contribute to the maintenance of homeostasis in normal endometriumand that perturbation of IL-6 mediated responses may play arole in disorders of the endometrium such as endometrial cancerand endometriosis.     

Cell kinetic study of the endometrium by nonisotopic in situ hybridization for histone H3 messenger RNA and immunohistochemistry for Ki‐67 and for estrogen and progesterone receptors

We investigated the cell kinetics of the endometrium in hysterectomy specimens taken for leiomyoma from 22 women with regular ovulatory menstrual cycles. Formalin‐fixed, paraffin‐embedded tissue sections were examined for proliferating activity using histone H3 messenger RNA in situ hybridization (H3 mRNA‐ISH) and immunostaining for the Ki‐67 antigen. The relationship of the proliferative activity of endometrial cells to the immunohistochemical expression of the estrogen receptor (ER) and the progesterone receptor (PR) was also examined. During the menstrual cycle, H3 mRNA expression was observed in both the epithelial cells and the stromal cells of the endometrium. In the functional layer, the labeling indices for H3 mRNA (H3 mRNA‐LIs) in the epithelial cells peaked in the late proliferative phase, decreased sharply in the early secretory phase, and remained unchanged thereafter. On the other hand, H3 mRNA‐LIs of stromal cells displayed two peaks: one in the midproliferative phase and the other in the late secretory phase, the former peak being the greater. In the basal layer, epithelial cells and stromal cells showed low H3 mRNA‐LIs and no significant variation throughout the menstrual cycle. The H3 mRNA‐LIs correlated well with the Ki‐67‐LIs and were lower than the corresponding Ki‐67‐LIs. The regression coefficient (H3 mRNA‐LIs against the Ki‐67‐LIs) was 0.33 for epithelial cells and 0.49 for stromal cells, suggesting that the cell cycle time was longer for epithelial cells than for stromal cells. The proliferative activity of endometrial cells showed close relationships with the expressions of ER and PR in the endometrium. When used in combination with other proliferative markers in paraffin‐embedded tissue sections, H3 mRNA‐ISH could open broader perspectives on the cell kinetics of the endometrium. Anat Rec 266:234–240, 2002. © 2002 Wiley‐Liss, Inc.     

Altered expression of HOXA10 in endometriosis: potential role in decidualization

Endometriosis is a poorly understood gynaecologic disorder thatis associated with infertility. In this study, we examined theexpression of HOXA10 in the eutopic endometrium of baboons withinduced endometriosis. A decrease in HOXA10 mRNA was observedafter 3, 6, 12 and 16 months of disease, which reached statisticalsignificance at 12 and 16 months. HOXA10 protein levels weredecreased in both the epithelial and stromal cells of the endometrium.Furthermore, expression of ß3 integrin (ITGB3), whichis upregulated by HOXA10, was decreased, whereas EMX2, a genethat is inhibited by HOXA10, was increased. Next, methylationpatterns of the HOXA10 gene were analysed in the diseased andcontrol animals. The F1 region on the promoter was found tobe the most significantly methylated in the endometriosis animalsand this may account for the decrease in HOXA10 expression.Finally, we demonstrate that stromal cells from the eutopicendometrium of baboons with endometriosis expressed significantlyhigher levels of insulin-like growth factor binding protein-1(IGFBP1) mRNA than disease-free animals in response to estradiol,medroxyprogesterone acetate and dibutyryl cAMP (H + dbcAMP).The functional role of HOXA10 in IGFBP1 expression was furtherexplored using human endometrial stromal cells (HSC). Overexpressionof HOXA10 in HSC resulted in a decrease of IGFBP1 mRNA, whereassilencing HOXA10 caused an increase of IGFBP1 mRNA, even inthe presence of H + dbcAMP. These data demonstrate that HOXA10negatively influences IGFBP1 expression in decidualizing cells.Thus, the decrease in HOXA10 levels may in part be involvedwith the altered uterine environment associated with endometriosis.     

Establishment of human endometrial cell cultures

Epithelial and stromal endometrial cells from 19 patients atdifferent phases of the menstrual cycle were enzymatically separated,isolated by successive centrifugation and primary cultures establishedfor in-vitro studies on implantation. The behaviour of cellsin vitro was evaluated using Nomarski's inverted optics, May-Grunwald-Giemsastained coverslips and scanning electron microscopy. Epithelialand stromal cells from all patients grew successfully in Chang'smedium and formed a mixed confluent monolayer of epithelloidand fibroblastic cells in 3–7 days and such monolayerscould be maintained alive up to 3–4 weeks. Epithelioidcells were polyhedral and grew as islands in a whorl-like wavypattern around glandular fragments. Fibroblasts were spindleshaped,more long-lived and grew rapidly to form parallel bundles ofcells. Significant differences were observed in the number ofmultinucleated cells and cells with intracytoplasmic vacuolesbetween endometrium from proliferative, postovulatory and secretoryphases (P < 0.01). Scanning electron mlcrographs showed cellswith cilia with varying densities of microvilli and apical protrusions.Endometrial cells in culture showed structural features remarkablysimilar to those described for cells in situ. The method describedallows the propagation in vitro of separate endometrium celltypes which can be used to study implantation mechanisms inunstiniu lated and stimulated cycles.     

Differential expression of angiopoietins 1 and 2 and their receptor Tie-2 in human endometrium

Angiogenesis, the growth of new capillaries from pre-existing blood vessels, is a physiological process involved in both normal menstrual cycling and implantation of the embryo. So far, very little is known about the expression of angiopoietins, growth factors involved in angiogenesis, in human endometrium. Both angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are ligands for the endothelial cell-specific receptor tyrosine kinase Tie-2. In this study we determined the mRNA expression of Ang-1, Ang-2 and Tie-2 by quantitative competitive RT/(QC)-PCR (including specifically designed competitor cDNA) in biopsied human endometrium throughout the menstrual cycle. We detected the mRNA for the angiopoietins in 30 out of 32 endometrial biopsies (94%), covering early proliferative (n = 4), mid proliferative (n = 12), late proliferative (n = 3), early secretory (n = 3), mid secretory (n = 5) and late secretory (n = 3) phases. Analysis of the target/competitor ratios (QC-PCR) revealed that Ang-1 mRNA expression was significantly up-regulated (P = 0.027) during the secretory phase of the menstrual cycle. In contrast, the expression levels of both Ang-2 mRNA and Tie-2 mRNA showed only minor variations at different cycle stages. These findings were confirmed by the relative expression ratio of Ang-1 versus Ang-2 in a multiplex PCR. The expression of Ang-1, Ang-2 and Tie-2 mRNA was detected in both isolated endometrial epithelial and stromal cell fractions. Immunohistochemical localization of the proteins revealed qualitative differences in both cell type and cycle stage expression. In conclusion, the enhanced Ang-1 expression during the secretory phase might serve to stabilize the newly developed blood vessels.     

Immunology: Investigations on the cell type responsible for the endometrial secretion of complement component 3 (C3)

It has been shown that and human endometria have the capacityto produce complement component 3 (C3). In rats, endometrialC3 is an oestrogen-dependent protein produced and secreted byglandular cells. The cell responsible for the synthesis andsecretion of human endometrial C3 has not been clearly defined.Our study was aimed at answering this question. Samples of endometriumobtained from hysterectomies were either immunostained for C3or digested with collagenase; then the stromal and glandularcells were separated and immunopurified (or not) with an antibodyto CD45 coupled to magnetic beads to eliminate the endometriallymphomyeloid cells. Cells were cultured for 2 weeks and C3measured in the medium by an in-house radioimmunoassay. Glandularas well as stromal cells stained positively for C3 and releasedC3 in vitro. The release of C3 from both cell types could beinhibited by cycloheximide. Epithelial cells produced significantlymore C3 than stromal cells, and endometrial C3 production washigher for both cell types when these were obtained from secretoryas compared to proliferative endometria. Lymphomyeloid cellswere possibly a source of C3 since after immunoadsorption ofthese cells, the remaining stromal or glandular cells producedsignificantly less C3. We conclude that endometrial stromal,glandular and lymphomyeloid cells all produce C3.     

Potential role for ET-2 acting through ETA receptors in experimental colitis in mice

Objective and design

This study attempted to clarify the roles of endothelins and mechanisms associated with ET_A/ET_B receptors in mouse models of colitis.

Materials and methods

Colitis was induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS, 1.5 mg/animal) or dextran sulfate sodium (DSS, 3%). After colitis establishment, mice received Atrasentan (ET_A receptor antagonist, 10 mg/kg), A-192621 (ET_B receptor antagonist, 20 mg/kg) or Dexamethasone (1 mg/kg) and several inflammatory parameters were assessed, as well as mRNA levels for ET-1, ET-2 and ET receptors.

Results

Atrasentan treatment ameliorates TNBS- and DSS-induced colitis. In the TNBS model was observed reduction in macroscopic and microscopic score, colon weight, neutrophil influx, IL-1β, MIP-2 and keratinocyte chemoattractant (KC) levels, inhibition of adhesion molecules expression and restoration of IL-10 levels. However, A192621 treatment did not modify any parameter. ET-1 and ET-2 mRNA was decreased 24 h, but ET-2 mRNA was markedly increased at 48 h after TNBS. ET-2 was able to potentiate LPS-induced KC production in vitro. ET_A and ET_B receptors mRNA were increased at 24, 48 and 72 h after colitis induction.

Conclusions

Atrasentan treatment was effective in reducing the severity of colitis in DSS- and TNBS-treated mice, suggesting that ET_A receptors might be a potential target for inflammatory bowel diseases.