Abnormal activation of Ras/Raf/MAPK and RhoA/ROCKII signalling pathways in eutopic endometrial stromal cells of patients with endometriosis.

BACKGROUND Enhanced proliferation and survival of eutopic endometrial cells from patients with endometriosis compared with healthy women is associated with abnormal activation of extra-cellular signal-regulated kinases 1 and 2 (ERK1/2). Given the role of Ras/Raf/mitogen-activated protein kinase (MAPK) and RhoA/ROCKII signalling pathways in the regulation of cell proliferation and migration, we analysed their possible roles in endometriosis. METHODS Primary eutopic endometrial stromal cells of patients with endometriosis (Eu-hESC, n= 16) and endometriosis-free controls (Co-hESC, n= 14) were harvested and subjected to proliferation and migration assays as well as kinase activity assays and immunoblot analysis of proteins from the Ras/Raf/MAPK and RhoA/ROCKII signalling pathways. Effects of ROCKII (Y-27632) and MAPK (U0126) inhibitors or siRNA knockdown of ROCKII, Raf-1 and B-Raf were analysed. RESULTS The proliferation rate of Eu-hESC was 54% higher than Co-hESC. Eu-hESC also displayed a 75% higher migration rate than Co-hESC. Eu-hESC displayed higher levels of ERK phosphorylation (83%) and p27 expression (61%) and lower levels of Raf-1 protein (47%) compared with controls. In addition to an inhibitory effect on cell proliferation, ROCKII knockdown led to significant down-regulation of cyclinD1 and p27 but did not affect ERK phosphorylation. Down-regulation of Raf-1 by siRNA was dispensable for cell proliferation control but led to an increase in ROCKII activity and a decrease in cell migration. B-Raf was shown to act as a regulator of hESC proliferation by modulating cellular ERK1/2 activity and cyclinD1 levels. Eu-hESC displayed 2.4-fold higher B-Raf activity compared with Co-hESC and therefore exhibit abnormally activated Ras/Raf/MAPK signalling. CONCLUSIONS We show that the same molecular mechanisms operate in Co- and Eu-hESC. The differences in cell proliferation and migration between both cell types are likely due to increased activation of Ras/Raf/MAPK and RhoA/ROCKII signalling pathways in cells from endometriosis patients.

A predictive model for endometriosis.

BACKGROUND: Aromatase is the key enzyme in the process of estrogen biosynthesis from the precursor androgen. Recently, aromatase has been found to be aberrantly expressed in eutopic endometrium of patients suffering from endometriosis. This finding has prompted speculation about the contribution of this enzyme to the prediction of this disease. METHODS: We prospectively aimed to evaluate whether endometrial biopsy, prior to laparoscopy in symptomatic women to screen for the presence of aromatase by real-time RT-PCR and immunohistochemistry, combined with select patients' characteristics, is of value to predict endometriosis. RESULTS: Of 48 consecutive symptomatic and eligible patients, 25 (52.1%) exhibited endometriosis and 23 (47.9%) were disease-free. A multiple logistic regression model revealed that 95.5% of patients whose eutopic endometrium was found to be positive for aromatase mRNA as well as immunohistochemically detected protein and who were additionally suffering from moderate to severe dysmenorrhoea (visual analogue scale score >4/10) exhibited endometriosis at laparoscopy. CONCLUSIONS: These findings provide direct evidence that screening for eutopic endometrial aromatase in combination with clinical data could be of discriminative value in the prediction of disease.

Interleukin-1 system and sex steroid receptor gene expression in human endometrial cancer.

OBJECTIVES: The interleukin-1 system is known to play a pivotal role in human physiology and reproduction. In the cycling endometrium, interleukin-1alpha activity is controlled by sex steroids and is confined to the perimenstrual phase, where it is involved in the events leading to tissue lysis and menstruation. Since local tissue degradation is also a feature of malignant tumors, our goal was to analyze the gene expression of interleukin-1alpha and other interleukin-1 family members and compare it with estrogen receptor alpha, estrogen receptor beta, and progesterone receptor mRNA expression in 27 endometrial carcinomas and 13 normal endometria. METHODS: Endometrial tumor tissues were obtained during hysterectomy for endometrial cancer, and normal endometrium was sampled in women undergoing surgical procedures for nonendometrial pathologies. Gene expression was analyzed by reverse transcription polymerase chain reaction. Protein expression was detected and localized by immunohistochemical staining. RESULTS: A strong gene expression of interleukin-1 type I receptor, estrogen recptor alpha, and progesterone receptor was detected in all tumor tissues and in the majority of benign endometrial tissues. However, in contrast to nonmalignant endometria, variable amounts of interleukin-1beta and interleukin-1 receptor antagonist mRNA were also detected in most of the tumor samples. Gene expression of interleukin-1alpha and estrogen receptor beta was considerably less frequent, with interleukin-1alpha being absent in all peri- and postmenopausal endometria and in all but one of the well-differentiated tumors. With decreasing differentiation interleukin-1alpha gene expression became more frequent. In these cases, interleukin-1alpha protein was detected predominantly in epithelial tumor cells of lower-grade tumors. CONCLUSION: We have demonstrated the presence of the interleukin-1 system in endometrial malignancies, and found a negative correlation between interleukin-1alpha and tumor differentiation. We hypothesize that the nonphysiological expression of interleukin-1alpha in less differentiated tumors might contribute to their invasiveness and malignant behavior.

Endometrial nuclear receptor co-factors SRC-1 and N-CoR are increased in human endometrium during menstruation.

Steroid hormone receptor co-factors are abundantly expressed in the uterus in order to modify steroid hormone receptor action, either leading to activation or repression of transcription in the endometrium. However, the role of co-factors in remodelling of the human endometrium has not been established. We therefore endeavoured to evaluate the presence of the co-activator SRC (steroid receptor co-activator)-1 and the co-repressors N-CoR (nuclear receptor co-repressor) and steroid co-repressor SMRT (silencing mediator of retinod and thyroid) receptors in the human endometrium during the different phases of the menstrual cycle. By using a real-time RT-PCR assay, we showed that SRC-1, N-CoR and SMRT mRNA are expressed in human endometrium during all phases of the menstrual cycle, as well as in inactive endometrium. Moreover, endometrial expression of SRC-1 and N-CoR mRNA increased during menstruation when compared with the other phases of the menstrual cycle (P < 0.001). Immunohistochemistry demonstrated that SRC-1 and N-CoR stain positive in the glandular epithelium and stroma in menstrual phase endometrium. The staining was weak in proliferative and secretory endometrium and absent in inactive endometrium. Our results suggest that differential expression of endometrial steroid receptor co-factors probably play a role in the regulation of human endometrium remodelling.

Calculated background elimination in quantifying nitric-oxide synthase enzyme activity.

Measuring nitric-oxide synthase (NOS) activity by monitoring the conversion of L-arginine to L-citrulline is currently the standard assay for NOS activity. We describe a simple method of quantifying low values of NOS activity by removing the background mathematically. When performing NOS activity studies in samples with low protein amount (< 25 microg/microl), we encountered the problem of sample values that can hardly be differentiated from blank values probably originating from radioactive-labeled arginine in the final eluate. Our method determines mathematically these background values and may be an improvement of the citrulline assay.

Estrogen increases endothelial carbon monoxide, heme oxygenase 2, and carbon monoxide-derived cGMP by a receptor-mediated system.

Carbon monoxide, a gaseous activator of soluble guanylyl cyclase formed by a subtype of the enzyme heme oxygenase designated heme oxygenase-2 in vascular endothelium, has been found to dilate blood vessels independently from nitric oxide. Because of the parallels between nitric oxide and carbon monoxide, we speculated that estrogen might affect carbon monoxide production in vascular endothelium. Endothelial cells of human origin (umbilical vein and uterine artery) were incubated for 4 or 24 h with 10(-12)-10(-6) M 17beta-estradiol. 17beta-Estradiol, at a concentration such as that attained during the ovulatory phase of the menstrual cycle (10(-10) M), administrated for 4 h led to a 2-fold increase in intracellular carbon monoxide production and heme oxygenase-2 protein levels (P < 0.05). A reporter assay, measuring the formation of cGMP as the direct product of carbon monoxide-induced activation of soluble guanylyl cyclase in endothelial cells, also revealed a 56% increase in cellular cGMP after treatment with 10(-10) M E2 17beta-estradiol (P < 0.05). By contrast, higher 17beta-estradiol concentrations had no significant respective effects due to nitric oxide synthase inhibition of carbon monoxide release. This 17beta-estradiol effect appeared to be ER dependent, as preincubation with tamoxifen (10(-6) M) blocked the stimulatory effect of 17beta-estradiol in each instance. Our preliminary data indicate a potential role for carbon monoxide as a biological messenger molecule in estrogen-mediated regulation of vascular tone.

Expression of inducible nitric oxide synthase in human breast cancer depends on tumor grade.

Expression of inducible nitric oxide synthase (iNOS) by tumor cells has been suggested to abrogate metastasis in several tumor models, whereas constitutive NOS expression correlated positively with tumor grade in human breast carcinoma. Whether or not expression of one of the various NOS isoforms could predict the prognosis of breast cancer, however, has not been established. In the present report we investigated the cellular distribution of NOS isoforms in a series of benign and malignant breast tumors and in normal breast tissue. Immunohistochemistry revealed that in samples of benign disease the number of iNOS+ epithelial cells or total epithelial cells was 69+/-16% (n = 50). In samples of grade II invasive ductal breast carcinomas the number of iNOS+ tumor cells or total tumor cells was 62+/-20% (n = 40), compared to 12+/-9% (n = 40) in samples of grade III carcinomas (P<0.0001). iNOS protein was also identifiable in most of the epithelial cells of normal breast tissue (n = 4). In contrast, eNOS protein was restricted to vascular endothelial cells in all of the specimens studied. Since the presence of tumor cell iNOS protein is inversely related to the tumor's metastatic potential, we conclude that endogenous tumor cell mediated iNOS expression might have an inhibitory effect on the metastatic process in breast cancer.

Human cervical ripening is associated with an increase in cervical inducible nitric oxide synthase expression.

The mechanisms that ultimately regulate cervical ripening during parturition remain largely unknown. A possible role for nitric oxide (NO) has recently emerged; however, the expression of NO synthase (NOS) within the human cervix in the ripening process has not been investigated. The purpose of this study was to identify cell types in the human cervix that contain NOS isoforms and to examine changes in their expression during the ripening process and the nonpregnant state. Inducible NOS (iNOS) immunoreactivity was observed in the epithelial cells and stromal spindle cells in 17 of 20 biopsies from cervices obtained within 10 min postpartum, but in only 4 of 12 nonpregnant controls (p = 0.03). Endothelial NOS (eNOS) immunoreactivity was restricted to vascular endothelia in all sections, whereas neuronal NOS was not detectable. Inducible NOS activity in the postpartum group was 3.2 times that of the control group (p = 0.0005), whereas constitutive NOS activity remained unchanged in both groups (p = 0.222). Competitive reverse transcription-polymerase chain reaction revealed no differences in the expression of iNOS (p = 0.443) or eNOS mRNA (p = 0.409). The existence of iNOS in the human postpartum cervix suggests that increased production of NO, probably induced by cytokines, may be relevant to the process of natural cervical ripening in humans.

Inducible nitric oxide synthase (iNOS) expression may predict distant metastasis in human melanoma.

Expression of inducible nitric oxide synthase (iNOS) and its cellular localization was investigated in subcutaneous or lymph node metastases of human melanoma. Immunohistochemistry revealed that iNOS expression was limited to melanoma cells. In samples of patients without distant metastases, the number of iNOS+ tumour cells/total tumour cells was 55% +/- 17% (n = 12) compared with 9% +/- 8% when distant metastases of lung, liver or brain occurred within an observation period of 3 years (n = 10) (P < 0.001). Western blotting confirmed the expression of iNOS protein in select cases. Notably, iNOS is expressed in regional melanoma metastases and its expression is inversely related to the tumour's metastatic potential. Thus, iNOS expression may have predictive value for the development of distant metastases of human melanoma.

Elevation of inducible nitric oxide synthase activity in human endometrium during menstruation.

Nitric oxide (NO) is a known agonist of programmed cell death (apoptosis). In order to discover its potential role during menstrual shedding, a process associated with extensive apoptosis, we evaluated activity and mRNA levels of the inducible and constitutive isoforms of NO synthase (NOS) in endometrial specimens of the proliferative (n = 11), late-secretory (n = 7), and menstrual (n = 17) phase of the cycle. These levels were compared with the proportion of apoptotic cells by detection of histochemically labeled DNA fragments. Inducible NOS (iNOS) activity during menstruation was six times that of the proliferative or late-secretory phase (p < 0.05), whereas constitutive NOS activity remained unchanged. Competitive reverse transcription-polymerase chain reaction revealed 146% and 77% increases of iNOS mRNA expression in the late-secretory and menstrual phases, respectively, compared to the proliferative phase (p < 0.05), whereas constitutive NOS mRNA expression remained constant. Inducible NOS immunostaining was restricted to epithelial cells, whereas constitutive NOS immunostainig was confined to vascular endothelia. In addition, the proportion of apoptotic cells within the glands of late-secretory or menstrual endometrium was twice that of the proliferative phase (p < 0.05). We conclude that local production of NO is involved in the signal transduction mechanisms leading to endometrial breakdown during menstruation.

Expression of endothelial (type III) nitric oxide synthase in cytotrophoblastic cell lines: regulation by hypoxia and inflammatory cytokines.

Expression of endothelial nitric oxide synthase (eNOS) has been localized to the villous syncytiotrophoblasts suggesting that NO release from these cells could prevent platelet adhesion and aggregation in the intervillous space. Hypoxia- or inflammation-dependent changes in the release of this vasoactive substance may result in thrombus formation and altered vascular resistance which occur in the placental bed of pre-eclamptic patients. To evaluate the influence of low-oxygen tension and inflammation on eNOS production in the trophoblast steady-state eNOS mRNA and protein levels were investigated in cytotrophoblastic BeWo and Jeg-3 cells cultured at 3.5 per cent oxygen and/or in the presence of the pro-inflammatory cytokines IL-1 and TNF-alpha. By RT-PCR and immunocytochemistry we demonstrate that BeWo cells produce eNOS mRNA and protein while eNOS polypeptide was undetectable in JEG-3 cells. In BeWo cells addition of both cytokines decreases eNOS mRNA and protein abundancies within 24 h of incubation while each substance alone had no effect. Compared to controls, the amount of eNOS transcripts was found to be elevated at low-oxygen tension, however, cNOS protein was downregulated after 24 h in the hypoxic environment, as shown by immunocytochemistry and Western blot analysis. Forskolin and methotrexate, which induce biochemical differentiation/ growth arrest in choriocarcinoma cells, stimulate eNOS mRNA and protein synthesis, but cannot overcome the decline of eNOS polypeptide levels during hypoxic incubation. It is speculated that acute hypoxia and inflammation impair eNOS/NO production of the trophoblast in vivo, which might contribute to pathological conditions of gestational diseases.

The effect of hormone replacement therapy on carotid arteries: measurement with a high frequency ultrasound system.

OBJECTIVE: To evaluate the effect of hormone replacement therapy (HRT) on carotid arteries in postmenopausal women with a high frequency ultrasound system. METHODS: In a clinical cross-sectional study carotid artery layers were measured in 82 postmenopausal women receiving a sequential regimen of HRT (oestradiol valerate 2 mg and dydrogesterone 10 mg) and in 70 postmenopausal women without HRT. Measurements of the left carotid artery layers (externa, media, intima) were taken with a single mechanically activated 22.5-MHz transducer with an effective band width of 8 MHz. RESULTS: A statistically significant increase in thickness of the media layer of the carotid artery was observed in the HRT group (0.34 +/- 0.06 mm) as compared to the untreated group (0.27 +/- 0.03 mm). The media/intima ratio of the treated group was statistically significantly higher than that of the untreated group (P < 0.05). The mean strength of the carotid wall was 0.70 +/- 0.17 mm in the 70 postmenopausal women without HRT and 0.76 +/- 0.24 mm in the 82 patients undergoing HRT. CONCLUSION: HRT has a morphological effect on the carotid arteries in postmenopausal women. These findings support a cardioprotective effect, especially in terms of prevention of atherosclerosis. This effect can be measured non-invasively by high frequency ultrasound.

Nitric oxide synthases in Kaposi's sarcoma are expressed predominantly by vessels and tissue macrophages.

Kaposi's sarcoma (KS) is a tumor of presumed vascular origin frequently found in patients with AIDS. Recent data suggest that the development of KS is linked with the presence of a newly recognized herpesvirus, human herpesvirus type 8. Nitric oxide (NO), a messenger molecule with vasoactive, antitumor, and antimicrobial effects, is produced by three isoforms of nitric oxide synthases (NOS). In the present report, we investigated the expression of NOS isoforms in KS. By NADPH-diaphorase histochemistry, NOS activity was detectable in endothelia and CD45+ cells within KS lesions. Reactivity for endothelial NOS (eNOS) was found in blood vessel endothelia; however, eNOS reactivity was negative in KS spindle cells in 12 of 17 tumors, and moderately positive in the other 5 lesions. In contrast to KS, tumor cells in three hemangiomas and one angiosarcoma were strongly positive for eNOS. Inducible NOS (iNOS) was absent from KS tumor cells but was found regularly in CD45+, HLA-DR+ cells within the lesions. In five KS-derived spindle cell cultures, neither eNOS nor iNOS proteins were detectable. The sporadic expression of eNOS by KS spindle cells in vivo and the absence of eNOS protein from KS spindle cells in tissue cultures argue against the possibility that the cells are derived from blood vessel endothelia. The consistent expression of iNOS by CD45+, HLA-DR+ cells within KS lesions strongly suggests that leukocyte-derived NO participates in the pathology of this tumor.

[The role of nitric oxide in reproduction]. / Die Rolle des Stickstoffmonoxids bei der Reproduktion.

Nitrix oxide (NO) is a highly reactive and short-lived radical (half-life time: 10-12 s), which is derived from L-arginine by the NO synthases (NOS) in several organ systems. The release of NO by endothelial cells leads to rapid relaxation of vascular smooth muscle cells, whereas release by several neuronal cells causes neurotransmission. When NOS is actively induced in immune cells or certain epithelia it causes cytotoxicity and/or apoptosis of these cells. In the reproductive organs NO is now considered to be an important trigger molecule for several physiological mechanisms. Follicular synthesized NO is involved in rupture of the follicle during ovulation. Moreover, NO participates in the acrosome reaction of spermatozoa during capacitation. Apoptosis and collagenolysis of the functional endometrium may be involved in endometrial shedding during menstruation. Since NO induces both apoptosis and collagenolysis, the newly discovered production of NO in late secretory endometrium could act as a key mechanism in the process of menstrual disintegration of the endometrium. Additionally, NO is necessary to support and maintain the decidualization process and plays a pivotal role in implantation.

Induction of inducible nitric oxide synthase expression in human secretory endometrium.

The endometrial secretory phase is characterized by stromal oedema, a premenstrual increase in stromal macrophages and an increased cytokine production as menstruation approaches. Nitric oxide (NO) is a mediator of vasodilatation and cytotoxicity which is synthesized from L-arginine by NO synthases (NOS). These enzymes are either constitutively expressed or induced by lipopolysaccharides and/or cytokines. The presence and function of the inducible isoform of NOS (iNOS) in normal human endometrium has not been fully elucidated until recently. Frozen tissue sections taken from 22 women who underwent hysterectomy and adnexectomy for benign disease were immunostained with antibodies raised against the different NOS isoforms to investigate the presence of NOS in human endometrium. iNOS stained positive in the glandular epithelial cells of the secretory endometrium. Staining was either weak or absent in the proliferative and inactive endometrium, as well as in the oviduct and the glandular epithelium of the endocervix. The stroma remained uniformly negative. Immunoreactivity for endothelial constitutive NOS (eNOS) was confined exclusively to endothelial cells. Furthermore, epithelial cells from endometrium, oviduct and endocervix and all endothelial cells showed positive staining for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, which is a histochemical marker for NOS activity. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed in order to assess the presence of NOS mRNA. Abundant expression of iNOS mRNA was detected in the secretory phase endometrium only. The strong expression of inducible NO synthase in human secretory phase endometrium suggests that the increased production of NO, probably induced by cytokines, may be relevant to the process of menstruation.

Estrogen does not induce the calcium-dependent nitric oxide synthase in cultured human uterine endothelial and myometrial smooth muscle cells.

In many tissues, estrogen-induced vasodilatation is mediated, at least in part, by the release of nitric oxide (NO). We determined whether human myometrial endothelial and smooth muscle cells express estrogen receptors (ERs) and whether endothelial NO synthase (eNOS) expression in these cells was affected by 17beta-estradiol (10[-13]-10[-6]M). ER was strongly expressed in myometrial smooth muscle cells but was absent from endothelial cells. Expression of eNOS mRNA was strong in endothelial cells, but weak in muscle cells. 17beta-estradiol administration for 24 or 72 h failed to increase eNOS in both cell types. Thus, an increase of human uterine blood flow by estrogens appears not to be mediated by stimulation of myometrial eNOS expression.

[Progesterone and nitric oxide systems]. / Progesteron und die Stickstoffmonoxidsysteme.

Sexual steroids play an established role in the mechanisms concerning reproduction, ovulation, menstruation and onset of labour. However, sexual steroids are also involved in extragenital mechanisms, which were described for both oestrogen, and progesterone. Progesterone and its mechanisms of signal transduction still remain to be fully understood. However, there is evidence, that nitric oxide (NO) seems to be an important mediator in these mechanisms. NO, the molecule, which was described to exist in acid rain, was elected the molecule of the year 1992 from the American Academy of Science. NO is a short-lived molecule, which is involved in many reactions as an modulating transmitter due to its high diffusibility and its polarity. NO regulates the immune response of mononuclear cells, contractility of smooth muscle cells and neuronal transmission of non-adrenergic and non-cholinergic nerves. Additionally it was found, that NO may be important in the regulation of menstruation, the maintenance of uterine quiescence as well as the initiation of labour and the maturation of the uterine cervix.

Presence of endothelial calcium-dependent nitric oxide synthase in breast apocrine metaplasia.

Endothelial calcium-dependent nitric oxide (NO) synthase has been shown to be expressed in human malignant breast tumours, and its presence correlates with tumour grade. Moreover, NO, being synthesised in breast tumour cells, may increase tumour blood flow and promote angiogenesis. In view of these aspects, we have assessed the distribution of NO synthase within a series of benign breast tumours using a monoclonal antibody against human endothelial calcium-dependent NO synthase. Activity was predominantly localised in apocrine metaplastic cells of fibrocystic disease, as well as in endothelia throughout all tissue sections. Consistent with previous reports, no endothelial calcium-dependent NO synthase immunoreactivity was observed in poorly differentiated infiltrating duct carcinoma cells. In conclusion, expression of endothelial calcium-dependent NO synthase in human breast apocrine metaplasia may be of significance in view of the NO's vascular effects in benign breast disease.