Department of Molecular Pathology and Innovative Therapies, - Anatomy and Cell Biology - Polytechnic University of Marche Via Tronto 10 A, 60020 Ancona, ITALY
Abstract: OBJECTIVES: This study investigated the influence that Fas and Fas ligand gene polymorphisms might have on preeclampsia. The pathogenesis of preeclampsia is still enigmatic and several studies have proposed that it may, in part, be determined by genetic susceptibility. Therefore, the identification of a gene polymorphism associated with an increased risk of preeclampsia might well represent a useful tool in the identification of at risk pregnant women enabling the setup of preventive therapy. Apoptosis has also been implied in the pathogenesis of preeclampsia and since Fas and Fas ligand are the main apoptotic pathway members, they may represent candidate genes involved in the development of preeclampsia. A polymorphism at the 670 position (A-G) in the Fas gene has been found more frequently in Hungarian women with preeclampsia. STUDY DESIGN: The study cohort was a group of 50 women with preeclampsia and 142 healthy control subjects from the general Italian population. They were studied, by RFLP analysis, to validate the role that the 670 G Fas gene polymorphism plays in preeclampsia, and to evaluate the Fas ligand IVS2nt 124 G polymorphism. The Fisher's exact test was used to compute the statistical difference between groups. RESULTS: The presence of the 670 G Fas gene variant was observed in 42 preeclamptic patients (84%) and 96 members of the general population control group (67.6%) (p=0.029). Regarding the Fas ligand gene, the IVS2nt 124 G variant was present in 14 preeclamptic patients (28%) and in 47 of the general population control subjects (33.1%) (p=0.6). CONCLUSIONS: The present study validated the hypothesis that the Fas 670 G variant may have an influencing role in preeclampsia.
Abstract: BACKGROUND: Activins are growth factors acting on cell growth and differentiation. Activins are expressed in high grade breast tumors and they display an antiproliferative effect inducing G0/G1 cell cycle arrest in breast cancer cell lines. Follistatin and follistatin- related gene (FLRG) bind and neutralize activins. In order to establish if these activin binding proteins are involved in breast tumor progression, the present study evaluated follistatin and FLRG pattern of mRNA and protein expression in normal human breast tissue and in different breast proliferative diseases. METHODS: Paraffin embedded specimens of normal breast (NB - n = 8); florid hyperplasia without atypia (FH - n = 17); fibroadenoma (FIB - n = 17); ductal carcinoma in situ (DCIS - n = 10) and infiltrating ductal carcinoma (IDC - n = 15) were processed for follistatin and FLRG immunohistochemistry and in situ hybridization. The area and intensity of chromogen epithelial and stromal staining were analyzed semi-quantitatively. RESULTS: Follistatin and FLRG were expressed both in normal tissue and in all the breast diseases investigated. Follistatin staining was detected in the epithelial cytoplasm and nucleus in normal, benign and malignant breast tissue, with a stronger staining intensity in the peri-alveolar stromal cells of FIB at both mRNA and protein levels. Conversely, FLRG area and intensity of mRNA and protein staining were higher both in the cytoplasm and in the nucleus of IDC epithelial cells when compared to NB, while no significant changes in the stromal intensity were observed in all the proliferative diseases analyzed. CONCLUSION: The present findings suggest a role for follistatin in breast benign disease, particularly in FIB, where its expression was increased in stromal cells. The up regulation of FLRG in IDC suggests a role for this protein in the progression of breast malignancy. As activin displays an anti-proliferative effect in human mammary cells, the present findings indicate that an increased FST and FLRG expression in breast proliferative diseases might counteract the anti-proliferative effects of activin in human breast cancer.
Abstract: Myostatin, a member of the TGF-beta superfamily of proteins, is known to suppress skeletal muscle mass and myocyte proliferation. The muscular component of the uterus is the myometrium, a tissue that regulates its mass in response to different physiological conditions under the influence of sex steroids. Recently, our laboratory reported effects of activin-A, another TGF-beta family member, on signalling and proliferation of rat uterine explants and human myometrial cell lines in culture. Here, we explore the expression, actions, and regulation of myostatin in uterine smooth muscle. Myostatin mRNA was demonstrated to be expressed in a myometrial cell line, pregnant human myometrial 1 cell line (PHM1). Functional assays showed that myostatin induced phosphorylation of Smad-2 and reduced proliferation of PHM1 number in a time and dose-dependent manner. Furthermore, myostatin activated smad-2 specific signalling pathways in rat uterine explants. To expand on our in vitro findings, we found that myostatin is expressed in rat uterus and determined that myostatin mRNA expression varies as a function of the phase of the estrous cycle. Uterine levels of myostatin peaked during late estrus and were the lowest at proestrus. Ovariectomy increased myostatin expression; estrogen treatment strongly decreased myostatin levels, whereas progesterone weakly decreased myostatin expression. In conclusion, myometrial cells are myostatin sensitive, myostatin mRNA levels are modulated in vivo in rats during the estrous cycle, and in response to steroid deprivation and replacement.
Abstract: Activin is a pleiotropic growth factor with a broad pattern of tissue distribution that includes reproductive tissues. Although direct actions of activin have been described in gonadal and uterine tissues, actions in the myometrium have not been defined. In this study we have characterized the responsiveness of uterine tissue and myometrial cell lines to activin-A. Uterine tissue and two myometrial cell lines, PHM1 (pregnant human myometrial 1) and hTERT HM (telomerase reverse transcriptase-infected human myometrial) respond to activin-A as measured by phosphorylation of Smad-2. Those cell lines express a full complement of activin receptors, as well as activin beta(A) subunit and follistatin. Activin inhibited proliferation of PHM1 and human telomerase reverse transcriptase-infected human myometrial cell line cells, with more extensive growth inhibition observed in PHM1s. In PHM1s, activin-A decreased oxytocin receptor and HoxA-10 mRNA expression but did not alter total progesterone receptor, cyclooxygenase-2 (Cox-2), and connexin 43 mRNA expression levels. Furthermore, treatment of PHM1 myometrial cells with activin-A attenuated oxytocin and thromboxaneA2 induced intracellular Ca(2+) accumulation. In conclusion, myometrial cells are activin sensitive, and activin-A can regulate myometrial cell functions.
Abstract: The present study investigated whether trophoblast, decidua and fetal membranes express nerve growth factor (NGF) mRNA and peptide. Tissue specimens were collected in the first and third trimester of pregnancy from women undergoing voluntary pregnancy interruption (no.= 6; from 8 to 12 gestational weeks) and from women having an elective caesarean section at term (no.= 6; week 39-40 of pregnancy). Using reverse transcriptase-polymerase chain reaction (RT-PCR), trophoblast, amnion/chorion and maternal decidua showed the expression of NGF mRNA both in early gestation and at term. By immunohistochemistry, the immunoreactive NGF was found in the cyto and syncytial trophoblast cells, chorionic mesodermic cells and in decidua. Vessel endothelial cells were stained in maternal compartments, while fetal vessels were unstained. These results, showing the expression and localization of NGF, support the current concept that human placenta is a potent neuroendocrine organ throughout gestation.
Abstract: OBJECTIVE: In the present study we evaluated the protein distribution and mRNA levels of inhibin alpha-subunit and its coreceptor betaglycan in endometrial adenocarcinoma. DESIGN: Two groups of postmenopausal women were studied: the first group had recently diagnosed endometrial adenocarcinoma (n = 16; age range 61-79 years), and the second group (n = 12; age range 64-78 years) had undergone hysterectomy for uterine prolapse and served as control. METHODS: Inhibin alpha-subunit and betaglycan gene expression and tissue distribution were evaluated by semiquantitative RT-PCR and immunohistochemistry respectively. RESULTS: Inhibin alpha-subunit and betaglycan mRNAs were expressed by both healthy and tumoral endometria, but their expression was significantly lower in endometrial carcinoma (P < 0.001, based on Student's t test). Inhibin alpha-subunit expression was much weaker in the glands of tumours than in non-neoplastic specimens. Betaglycan protein was identified in the epithelial cells lining non-tumoral endometrium, and in endothelial cells of both normal and tumoral endometria. Well-differentiated neoplastic cells had a faint and scarce betaglycan staining, and poorly differentiated cells did not express betaglycan at all. CONCLUSIONS: The lower inhibin alpha and betaglycan expression in endometrial adenocarcinoma suggests that the inhibin action may be disrupted. However, the expression of betaglycan in the endothelia of the tumour vasculature suggests that a selective vascular response to inhibin may be possible in these tumours.
Abstract: BACKGROUND: Preeclampsia (PE) is a disorder that occurs in at least 5% of pregnancies and affects both the mother and the unborn baby. A dramatic increase of maternal serum inhibin A concentration in the second and third trimester of pregnancy is a common feature of PE and inhibin A measurement may add significant prognostic information for predicting PE in pregnant women. DESIGN: We evaluated the presence and prevalence of gene polymorphisms for inhibin alpha subunit (INHalpha) in patients affected by PE (no.=50; study group), and in the general population (control group composed of 103 women and 42 men). METHODS: DNA extraction, single strand conformation polymorphism analysis, DNA sequencing, restriction fragment length polymorphism analysis, and Fisher's exact test were used. RESULTS: A 769G-->A transition was found in INHalpha1, but not in INHalpha2 or INHalpha3 fragment. This variant was found in 10/145 normal controls (7,6%), and in 1/50 preeclamptic patients (2%), without significant difference between the two groups (p=0.29). CONCLUSIONS: The prevalence of INHalpha gene variants is not increased in PE. Due to its frequency, the 769G-->A transition may be considered a polymorphism present in the general Italian population.
Abstract: OBJECTIVE: Activin A is a multifunctional growth and cell differentiation factor produced by normal endometrium, and secreted in high amounts by endometrial adenocarcinoma. In the present study we evaluated the expression of two inhibitory activin A ligands, follistatin and follistatin-related gene (FLRG), in endometrial adenocarcinoma and in age-matched healthy human endometrium. DESIGN AND METHODS: Atropic menopausal (n=13) and tumoral (n=9 adenocarcinoma) tissues were processed to evaluate mRNA expression levels (by semiquantitative RT-PCR) and peptide localization (by immunohistochemistry). Differences were evaluated by the unpaired t-test and assumed to be statistically significant when P<0.05. RESULTS: Both control and tumoral endometrial samples express and localize follistatin and FLRG. However, whereas follistatin mRNA expression did not differ significantly, FLRG was significantly lower in endometrial adenocarcinoma than in healthy endometrial specimens (P<0.0001). With respect to the localization of proteins, follistatin was immunolocalized in endometrial epithelial and vascular cells both in tumoral and healthy endometrium without any significant difference in intensity. Nuclear and cytoplasmic FLRG immunolocalization was seen in glands, and only nuclear immunolocalization was found in stroma and vessels of healthy endometrium. FLRG was weakly immunostained in endometrial adenocarcinoma. CONCLUSIONS: Whilst follistatin expression is unchanged, FLRG is down-regulated in endometrial carcinoma. As activin A is a differentiation factor of human endometrium, the present findings support an imbalance between increased activin A and decreased FLRG expression in endometrial cancer, so that the failure of the activin A pathway through FLRG may be pivotal in endometrial tumorigenesis.
Abstract: Human placenta, decidua, and fetal membranes are the major sites of production and secretion of inhibin A and activin A in maternal serum, amniotic fluid, and umbilical cord blood. These tissues also express follistatin-related gene and betaglycan, the binding proteins of activin A and inhibin A, respectively, recently identified. They show a different expression throughout pregnancy, suggesting new functional roles into gestational tissues. The availability of suitable assays for measuring inhibin A and activin A lead us the possibility to investigate their secretion in healthy pregnancy. In addition, several evidences underline the potential role and the clinical usefulness of their measurement in the diagnosis, prevention, prognosis and follow-up of different gestational pathologies such as: threatened abortion, placental tumors, hypertensive disorders of pregnancy, intrauterine growth restriction, fetal hypoxia. The measurement of inhibin A and activin A into the biological fluids of pregnancy will offer in the future further possibilities in early diagnosis, prediction, and monitoring pregnancy diseases.
Abstract: Activin-A is expressed by human endometrium, and the actions are counteracted by follistatin, its binding protein. We evaluated the endometrial mRNA and peptide expression of follistatin-related gene (FLRG), a protein that binds activin-A, preventing its interaction. By reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry, FLRG expression was evaluated in tissues collected at early proliferative (EP; n = 8) and late proliferative (LP; n = 8); early secretory (ES; n = 9) and late secretory (LS; n = 10); and in pregnancy, maternal decidua (MD; n = 12). FLRG mRNA was expressed by all samples, and semi-quantitative analysis showed that FLRG expression was significantly ( P < 0.001) higher in MD. FLRG was strongly immunolocalized in epithelial cells of glands and vessel walls (cytoplasma and nucleus), but only in the stromal cells nucleus. In MD, FLRG immunostaining was found in the nucleus and cytoplasm of vessel endothelium, gland epithelial, and decidualized stromal cells. In conclusion, FLRG is expressed by the human endometrium, and the different cellular localization suggests novel putative functions.
Abstract: Under the influence of ovarian steroid hormones, endometrial cells aer able to produce a wide variety of growth factors and peptide hormones that area believed to promote: (1) physiological growth and differentiation during the endometrial cycle; (2) decidualization, an essential preparative event for establishment of pregnancy; and (3) pathological growth and differentiation in endometriosis and cancer. Among the local factors produced by the human endometrium, corticotropin-releasing factor (CRF) and activin A have been evaluated in terms of localization and effects. CRF is a neuropeptide expressed by the epithelial and stromal cells of the human endometrium in increasing amounts from the endometrial proliferative to the secretory phase. CRF expression also increases in the pregnant endometrium, from early in the pregnancy until term. CRF-type 1 receptor mRNA is only expressed by stromal cells. Progesterone induces CRF gene expression and release from decidualized cells and CRF decidualizes cultured stromal endometrial cells. Urocortin, a CRF-related peptide, has been identified in endometrial epithelial and stromal cells, and its function is still under investigation. Activin A is a growth factor expressed in increasing amounts throughout endometrial phases by both epithelial and stromal cells. This growth factor is secreted into the uterine cavity with higher levels in the secretory phase. Maternal decidua expresses activin A mRNA in increasing amounts from early pregnancy until term. Human endometrium also expresses activin-A receptors and follistatin, its binding protein. Activin A decidualizes cultured human endometrial stromal cells (an effect reversed by follistatin) and modulates embryonic trophoblast differentiation and adhesion. Activin A is expressed in endometriosis and endometrial adenocarcinoma.
Abstract: BACKGROUND: Betaglycan is a membrane-anchored proteoglycan involved in mediating the passage of transforming growth factor-beta (TGF-beta), inhibin and activin activities into cells. TGF-beta and inhibin-related proteins are growth factors that are expressed by several tIssues and in pregnancy. They have a function in modulating the growth, differentiation and invasion of the placental trophoblast. OBJECTIVE: To evaluate whether betaglycan is expressed by intrauterine tissues throughout gestation. DESIGN AND METHODS: Expression of betaglycan mRNA and protein was evaluated (by RT-PCR and immunohistochemistry, respectively) in trophoblast, decidua and fetal membranes collected during the first (n=6 elective terminations of pregnancy, between 8 and 12 gestational weeks) and third (n=6 elective caesarean sections, between 39 and 40 weeks) trimesters of pregnancy. RESULTS: Betaglycan mRNA was expressed by all gestational tIssues, independently of gestational age. Immunoreactive protein was found in decidual cells and in some chorionic, but not epithelial, amniotic cells. With respect to the placental localization, syncytiotrophoblast, but not cytotrophoblast, cells were intensively stained both in the placental bed and in the villous trophoblast, and in some cells within the stroma of terminal villi, of the first and third trimesters of pregnancy. Immunoreactive betaglycan was demonstrated in the endothelial cells of decidual vessels in both the first and third trimesters of pregnancy, whereas endothelial cells of fetal blood vessels in the villous were clearly represented only in first trimester samples, not in those of term placenta. CONCLUSIONS: Betaglycan mRNA and peptide are expressed by the trophoblast, the decidua and the fetal membranes, but the localization of the peptide in vessel walls is dependent on gestational age.
Abstract: Activin A is a placental glycoprotein and possible biological actions during pregnancy, suggested by experimental data, are the modulation of cytotrophoblast differentiation, placental hormonogenesis and uterotonins secretion. Follistatin-related gene (FLRG) is a 70 amino acids protein which binds activin A with high affinity, and which modulates its biological effects on target tissues by preventing the activin A interaction with its receptors. The present study investigated whether trophoblast, decidua and fetal membranes express FLRG mRNA (by RT-PCR) and peptide (by immunohistochemistry). Tissue specimens were collected at first and third trimester of pregnancy, from patients undergoing voluntary pregnancy interruption (no.=6; from 8 to 12 gestational weeks) and elective caesarean section at term (no.=6; 39-40 weeks of pregnancy). FLRG mRNA was expressed by the various gestational tissues both at early gestation and at term pregnancy. Immunoreactive protein was found in the trophoblast cells, epithelial amniotic and chorionic cells and maternal decidua; nevertheless, the most intense FLRG stain was detected in the walls of decidual and placental blood vessels. In conclusion, FLRG mRNA and peptide are expressed by placenta and fetal membranes. Its different immunolocalization with respect to follistatin and activin A supports a different role for FLRG in modulating activin A actions into gestational tissues.
Abstract: OBJECTIVE: Activin A is a growth factor expressed by human endometrium, and its biologic effects are counteracted by follistatin. We evaluate whether activin A and follistatin mRNA and peptide expression as well as protein secretion from human endometrium change throughout the menstrual cycle. METHODS: In 25 healthy fertile patients, uterine washing fluid was retrieved by hydrosonography. In a subgroup (n = 13), endometrial tissue samples were collected by hysteroscopy during the proliferative (n = 6) or secretory (n = 7) phase of the menstrual cycle. Activin and follistatin mRNA and peptide expression were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and by immunohistochemistry (IHC), respectively. Activin A and follistatin levels were assayed in uterine washing fluids by specific enzyme-linked immunosorbent assays and evaluated according to the endometrial thickness and menstrual cycle days. RESULTS: Both activin A and follistatin mRNAs were expressed by human endometrium, and their peptides immunolocalized both in proliferative and secretory endometrial epithelial and stromal cells. A significant increase in immunoreactive activin betaA but not in follistatin was observed in glandular epithelium during the secretory phase. Activin A but not follistatin was significantly (P <.0001) higher in the washing fluids collected during the secretory than proliferative phase of the menstrual cycle. In addition, a significant correlation was found between activin A, but not follistatin, and menstrual cycle days (P <.0001) or endometrial thickness (P <.0001). CONCLUSIONS: Both activin A and follistatin mRNAs are expressed by human endometrium; however, activin A but not follistatin peptide expression and secretion were increased in the secretory phase of the menstrual cycle, suggesting an important role in human endometrium.
Abstract: PCR amplification of the complete intergenic spacer region (IGS) of the Tuber borchii nuclear ribosomal repeat was obtained using universal primers CNL 12 and NS1rev. In order to improve amplification yield a specific primer, T1, was selected from a partial sequence of the IGS product. IGS diversity was characterized both at the intraindividual and intraspecific level. The results obtained at the intraindividual level showed 10% varying repeats on ten screened colonies, while at the intraspecific level the IGS polymorphism was evident as difference in length amplification between mycelial strains and fruit bodies: 3.5 kb and 2 kb respectively.
Abstract: The sequence and characterisation of the entire nuclear rDNA intergenic spacer (IGS) for the genus Tuber are presented. Sequence analyses showed that the organisation of the Tuber borchii rDNA IGS is typical of rDNA spacers, consisting of a central repetitive region and flanking unique sequences on either side. Direct repeats, symmetry elements, tandem repeats and possible areas of recombination were found. The putative ends of the 25S and 17S rDNA were identified. The presence of 5S rDNA in the IGS region was excluded.
Abstract: Urocortin is a 40-amino acid peptide belonging to the corticotropin-releasing factor (CRF) family. In human reproductive tissues, urocortin expression has been previously demonstrated in the ovary, in the placenta and fetal membranes and in pregnant uterine tissues, while no data are available on the expression of the peptide in the nonpregnant uterus. In this study, urocortin expression was evaluated by both immunohistochemistry and reverse transcription-polymerase chain reaction, in human uterine tissues and cells at different phases of the menstrual cycle. Urocortin was immunolocalized in endometrial epithelial and stromal cells, as well as in the myometrium, and in vascular smooth muscle cells. No differences between proliferative and secretory phase were observed. These results were confirmed by reverse transcription-polymerase chain reaction analysis of isolated endometrial epithelial and stromal cells, and myometrial specimens. These findings open new questions on the roles played by urocortin in the human uterus.
Abstract: The placenta and its accessory membranes amnion and chorion undertake the role of intermediary barriers and active messengers in the maternal-fetal dialog. They synthesize, metabolize, and serve as target to numerous hormones that regulate maternal and fetal physiology during pregnancy. Among these factors, corticotropin-releasing factor (CRF) has been one of the more investigated in the last decade. Increasing evidence indicates that in the event of acute or chronic metabolic, physical, or infectious stress, maternal or fetal physiologic and pathologic conditions may influence placental secretion of CRF. The current opinion is that the placenta actually takes part in a stress syndrome by releasing CRF, which may help to influence uterine perfusion, maternal metabolism, fluid balance, and possibly uterine contractility, thereby protecting the fetus from a hostile environment.
Abstract: Activin A (beta A beta A) and inhibin A (alpha beta A) are dimeric glycoproteins secreted from early to term pregnancy in the maternal circulation. They circulate in higher amounts in women with gestational hypertension and/or pre-eclampsia, the most important gestational diseases also causing fetal growth restriction (FGR). Since no data are available in patients with pre-eclampsia and superimposed FGR, by using two-site immunoassays we evaluated serum activin A and inhibin A levels in serum samples collected from: healthy normotensive pregnant controls (n = 42); and women with pre-eclampsia with (n = 19) or without superimposed FGR (n = 21). In addition, by quantitative reverse transcriptase-polymerase chain reaction the changes of alpha- and beta A-subunit mRNA expression in placentas collected from healthy controls (n = 7) and pre-eclamptic pregnancies with (n = 6) or without (n = 6) superimposed FGR was also investigated. Activin A and inhibin A serum levels were significantly higher in pre-eclampsia, and the presence of FGR did not significantly modify these concentrations. Similarly, inhibin-subunit mRNA levels in placentas from pre-eclampsia were significantly higher than in controls, and FGR did not significantly affect this expression. The present data suggest that the increased placental expression of inhibin subunit mRNAs is part of the mechanism leading to increased serum activin A and inhibin A levels.
Abstract: Inhibin-related proteins are involved in the control of the feto-maternal communication required to maintain pregnancy. Human placenta, decidua, and fetal membranes are the major sites of production and secretion of activin A, inhibin A and inhibin B in maternal serum, amniotic fluid, and cord blood. The availability of suitable assays developed in the last years has enabled the measurement of inhibins and activin A in their dimeric forms, in order to investigate their role in physiological conditions of pregnancy. The studies conducted on inhibin-related proteins and human pregnancy suggested the possibility of an involvement of inhibin A and activin A in the pathogenesis of gestational diseases. In fact, several lines of evidence underline the potential role and the clinical usefulness of inhibin-related proteins measurement in the diagnosis, prevention, prognosis and follow-up of different gestational pathologies such as early pregnancy viability, Down's syndrome, fetal demise, pre-eclampsia, pregnancy-induced hypertension, preterm delivery and intrauterine growth restriction. The measurement of inhibin A and activin A into the biological fluids of pregnancy will offer in the future, further possibilities in the early diagnosis, prediction, and monitoring diseases of pregnancy.
