Walter Tschugguel

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.

Simultaneous expression of nitric oxide synthase and estrogen receptor in human breast cancer cell lines

SummaryFor various human tumor cell lines (neuroblastoma, cervix carcinoma) the presence of constitutive nitric oxide synthase (cNOS) has been documented. Here, for the first time, we report about cNOS expression in 10 of 16 human breast cancer cell lines. cNOS expression correlates strongly with expression of estrogen receptor (ER). Competitive reverse transcription — polymerase chain reaction (cRT-PCR) was used to detect cNOS and ER mRNA expression. Our findings suggest that estradiol could stimulate constitutive NO release in breast tissue, where it acts as a free radical.

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

SummaryExpression 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.

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.

Estrogen Receptor-β Is the Predominant Estrogen Receptor Subtype in Normal Human Synovia

Objective: Joint pain increases after menopause with more than 50% of woman suffering from arthralgies. Since pain and inflammation of joints originate from synovial tissue, we aimed to discover whether estrogen receptors are present in the human synovia. Methods: This in vitro study was performed on samples of human synovial tissue, obtained from pre-(n = 8) and postmenopausal woman (n = 11) and men (n = 5) following surgery due to traumatic lesions. Fresh synovial tissue specimens were assessed for the localization as well as the presence of estrogen receptor-α (ERα) and estrogen receptor-β (ERβ) by means of immunohistochemistry, as well as Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. Results: ERβ protein and mRNA were found to be equally and highly expressed in synovial stroma and lining cells of all explants independent of sex or menopausal status. In contrast, weak ERα staining was localized in the synovial lining cells in only three of 24 explants. ERα protein was found to be weakly expressed in three of ten explants. ERα mRNA was found with highly variable amounts in seven of ten explants. Conclusion: In view of our observation that ERβ but not ERα is expressed regularly in normal human synovia in high amounts, we propose that estrogen could play a significant role in synovial membrane function in women and men, operating preferably via the ERβ isoform.

High precision measurement of electrical resistance across endothelial cell monolayers.

Effects of vasoactive agonists on endothelial permeability was assessed by measurement of transendothelial electrical resistance (TEER) of human umbilical vein endothelial cells (HUVECs) grown on porous polycarbonate supports. Because of the low values of TEER obtained in this preparation (< 5 Ωcm2) a design of an Ussing type recording chamber was chosen that provided for a homogeneous electric Held across the monolayer and for proper correction of series resistances. Precision current pulses and appropriate rates of sampling and averaging of the voltage signal allowed for measurement of < 0.1 Ω resistance changes of the endothelium on top of a 21 Ω series resistance of the support and bathing fluid layers. Histamine (10 μM) and thrombin (10 U/ml) induced an abrupt and substantial decrease of TEER, bradykinin (1 μM) was less effective, PAF (380 nM) and LTC4 (1 μM) had no effect TEER was also reduced by the calcium ionophore A-23187 (10 μM). The technique allows for measurements of TEER in low resistance monolayer cultures with high precision and time resolution. The results obtained extend previous observations in providing quantitative data on the increase of permeability of HUVECs in response to vasoactive agonists.

Treatment of functional hypothalamic amenorrhea with hypnotherapy

OBJECTIVE To determine the effects of hypnotherapy on resumption of menstruation in patients with functional hypothalamic amenorrhea (FHA). DESIGN Uncontrolled clinical study. SETTING Academic clinical care center. PATIENT(S) Twelve consecutive women with FHA were selected. INTERVENTION(S) A single 45- to 70-minute session of hypnotherapy was administered, and patients were observed for 12 weeks. MAIN OUTCOME MEASURE(S) Patients were asked whether or not menstruation resumed and whether or not well-being and self-confidence changed. RESULT(S) Within 12 weeks, 9 out of 12 patients (75%) resumed menstruation. All of the patients, including those who did not menstruate, reported several beneficial side effects such as increased general well-being and increased self-confidence. CONCLUSION(S) Hypnotherapy could be an efficacious and time-saving treatment option that also avoids the pitfalls of pharmacological modalities for women with FHA.

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 NOs vascular effects in benign breast disease.

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.

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.