Expression patterns of two serine protease HtrA1 forms in human placentas complicated by preeclampsia with and without intrauterine growth restriction.

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are pregnancy-specific disorders that have in common abnormal placental implantation, a marked proliferation of villous cytotrophoblastic cells and focal necrosis of the syncytiotrophoblast. Several studies show an ischemic placenta with a high-resistance vasculature, which cannot deliver an adequate blood supply to the feto-placental unit. The cause of PE is a matter of debate, but recently studies in mice suggest that the primary feto-placental lesions are sufficient to initiate the disease. HtrA1, a member of the family of HtrA proteins, is a secreted multidomain protein with serine protease activity. It is expressed in first and third trimester of gestation. In specimens from the first trimester of gestation, immunostaining for HtrA1 is generally found in both layers of villous trophoblast, syncytiotrophoblast and cytotrophoblast. Cytoplasm of extravillous trophoblast and extracellular matrix of cell islands and cell columns are labeled for HtrA1. Specimens from third trimester of gestation show a more intense positivity for HtrA1 in the syncytiotrophoblast than in cytotrophoblast. The extravillous trophoblast and the decidual cells, is positive for HtrA1. The purpose of this study is to investigate the expression pattern of HtrA1 in placentas from PE without IUGR (maternal PE) and with IUGR (fetal PE) by quantitative western blotting and immunohistochemistry. By quantitative western blotting analysis we observed a significant upregulation of approximately 30 kDa HtrA1 form in PE. Differently, we detected a significant total HtrA1 down-regulation in PE-IUGR. Moreover, immunostaining for HtrA1 was positive in the villous trophoblast, in the syncytial knots and irregularly in the fetal vessel walls in PE placentas while immunostaining for HtrA1was present particularly in the syncytial knots in PE-IUGR placentas. In conclusion, we suggest that the approximately 30 kDa HtrA1 form can be correlated to maternal PE while that the significant down-regulation of total HtrA1 can be correlated to placental PE. These HtrA1 alterations could be considered as possible markers to discriminate placental PE from maternal PE.

Expression of basic fibroblast growth factor, its receptors and syndecans in bladder cancer.

Basic fibroblast growth factor (bFGF) is a heparin-binding cationic protein involved in a variety of pathological conditions including angiogenesis and solid tumour growth. The basic fibroblast growth factor receptor (FGFR) family comprises at least 4 high affinity tyrosine kinase receptors that require syndecans for their function. Mounting evidence indicates that syndecans, that bind both bFGF and their FGFRs, will act as stimulators, whereas syndecans that only bind bFGF will act as inhibitors of signaling by sequestering the growth factor. Recent findings have highlighted the importance of syndecans in urological cancers. The aim of this study is to investigate the expression of bFGF, its receptors (R1 and R2) and syndecans (1-4) in invasive urothelial carcinoma and normal-looking urothelium by Western blotting, RT-PCR, and immunohistochemistry analyses. Interestingly, bFGF, FGFR1 and FGFR2 protein levels statistically increased in bladder cancer tissues. mRNA of FGFR1 and syndecans (1-4), showed a statistically significant increase while an mRNA increase in the other molecules analysed was not significant. bFGF, its receptors and syndecan immunostaining were mainly present in the urothelium both in normal-looking tissues and urothelial neoplastic cells. In conclusion, our data report that the bFGF, FGFR and syndecan expressions are altered in bladder tumours.

Remodeling of uterine innervation.

This minireview reports current hypotheses concerning the remodeling of sympathetic innervation in rodent and human uterus during the estrous cycle and gestation. Neural modulation in this organ is related to sexual hormone concentrations, and a reduction in nerve density is observed when estrogen levels are high during the estrous cycle. Estrogen receptor alpha is considered to be the major receptor mediating the action of estrogen. In the uterus, the expression of neurotrophins, such as nerve growth factor, which are involved in the survival and growth of nerve fibers, changes in response to steroid levels. Despite much research, further studies are necessary to clarify various aspects of nerve growth control under diverse physiological conditions. These studies could be of importance, since alterations of the biological mechanisms of uterus innervation may play significant roles in various pathologies, such as infertility and spontaneous abortion.

Evidence for a role of TGF-beta1 in the expression and regulation of alpha-SMA in fetal growth restricted placentae.

There is evidence that alpha-smooth muscle actin (alpha-SMA) is a protein that plays a pivotal role in the production of contractile forces and it is induced by transforming growth factor-beta1 (TGF-beta1). We have analysed the expression of alpha-SMA, TGF-beta1, its receptor RI and the activator phospho-Smad2 in (a) fetal growth restriction pre-eclamptic placentae characterised by early onset and absence of end diastolic velocities in the umbilical arteries (FGR-AED) and (b) control placentae accurately matched for gestational age. The study was performed by immunohistochemical, quantitative Western blotting, ELISA, RT-PCR and in vitro analyses. We found that TGF-beta1 stimulates alpha-SMA production in chorionic villi cultured in vitro. In addition, we observed that in vivo TGF-beta1 concentration is significantly higher in FGR-AED placental samples than in control placentae and that this growth factor could have a paracrine action on villous stroma myofibroblasts expressing TGF-beta1 receptors and phospho-Smad2. Indeed, we report that alpha-SMA undergoes a redistribution in FGR-AED placental villous tree, i.e. we show that alpha-SMA is enhanced in medium and small stem villi and significantly decreased in the peripheral villi. Our data allow us to consider TGF-beta1 and alpha-SMA as key molecules related to FGR-AED placental villous tree phenotypic changes responsible for increased impedance to blood flow observable in this pathology.

The solitary chemosensory cells and the diffuse chemosensory system of the airway.

Solitary chemosensory cells (SCCs), which resemble taste bud cells, are present in the epidermis and oropharynx of most primary aquatic vertebrates. Recent studies have led to the description of SCCs also in mammals too. In the airway and digestive apparatus, these elements form a diffuse chemosensory system. SCCs do not aggregate into groups and in SCCs, as in taste bud cells, immunoreactivity forthe G-protein subunit alpha-gustducin and for other molecules of the chemoreceptive cascade was found. Questions remain about the role of the diffuse chemosensory system in control of complex functions (e.g. airway surface liquid secretion) and about the involvement of chemoreceptors in respiratory diseases. Therapeutic actions targeting chemoreceptors could be tested in the treatment of respiratory diseases.

High temperature requirement A1 and fibronectin: two possible players in placental tissue remodelling.

High temperature requirement A1 (HtrA1) is a secreted protease involved in placental development. Fibronectin (FN) is involved in important process such as wound healing, cell adhesion and spreading, growth, migration, and differentiation. The purpose of this study was to analyse the expression patterns of HtrA1 in relationship to FN and to the key growth zones of placenta such as mesenchymal villi as well as cell islands and cell columns. We demonstrated that FN and HtrA1 are localized in the placental key growth zones suggesting a pivotal role in maintaining the balance among the molecules involved in the placental development and differentiation.

Placental expression of substance P and vasoactive intestinal peptide: evidence for a local effect on hormone release.

The present study evaluated vasoactive intestinal peptide (VIP) and substance P (SP) mRNA expressions and the localization of both peptides in first- and third-trimester human placentas. VIP and SP mRNAs were detected by slot blot analysis in first- and third-trimester placental tissues. By immunohistochemistry both neuropeptides were localized in the trophoblast (syncytium and cytotrophoblastic cells) of the chorionic villi. Because little information is available on the role of VIP and/or SP on the secretion of placental hormones, we investigated the effect of these neuropeptides on human chorionic gonadotropin (hCG) and progesterone release from primary cultured human trophoblastic and JEG-3 cells. The addition of increasing doses of VIP resulted in a dose-dependent stimulation of hCG release from cultured human trophoblast and JEG-3 cells. Increasing doses of VIP also dose-dependently stimulated progesterone secretion from primary cultured trophoblastic cells at all time points evaluated and from JEG-3 cells only after 3 h. SP did not affect hCG and progesterone secretion either in cultured human trophoblast or in JEG-3 cells. In conclusion, the present study demonstrates that VIP and SP are mainly expressed in human trophoblasts, and that VIP modulates the in vitro secretion of hCG and progesterone, suggesting a different role in trophoblastic function of the two peptides.

High temperature requirement A1, transforming growth factor beta1, phosphoSmad2 and Ki67 in eutopic and ectopic endometrium of women with endometriosis.

Increasing evidence supports the hypothesis that TGFb1 signalling may be mediated by high temperature requirement A1 (HtrA1) serine protease, acting on important regulatory mechanisms such as cell proliferation and mobility. Evidence is now accumulating to suggest that HtrA1 is involved in the development and progression of several pathologies. The aim of this study was to evaluate: i) if HtrA1 and TGFb1 expressions differ in eutopic and ectopic endometrium in women with endometriosis; ii) if HtrA1 correlates to TGFb1, pSmad and Ki67. This study was carried out including 10 women with ovarian endometriosis (cases) and 10 women with non endometriotic diseases (controls). Endometrial tissue underwent immunohistochemical H-score analysis for HtrA1, TGFb1, pSmad and Ki67 molecules. Data evaluation was performed by a nonparametric Kruskal-Wallis test and Spearman correlation was applied to evaluate the relationship among the molecules investigated in the epithelial and in the stromal compartment. The HtrA1 was significant decreased in ectopic and eutopic endometrium of women with endometriosis when compared with control endometrium in epithelial compartment. TGFb1was significantly increased in eutopic endometrium and decreased in ectopic endometrium in epithelial and stromal compartment. In addition, Ki67 was significant increased and an increase, but not significant, was detected for pSMAd2 in eutopic and ectopic endometrium compared to control one.  In summary, the significant direct correlation between TGFb1 and pSmad2 as well as between HtrA1 and TGFb1 and the very significant increase of Ki67 in stromal compartment of eutopic endometrium suggest a possible involvement of HtrA1 in the pathogenesis of endometriosis.

Differentiation of human trophoblast populations involves alterations in cytokeratin patterns.

Cytokeratins (CKs) are related to proliferation and differentiation of epithelial cells. Little knowledge exists about CK patterns in human trophoblast subpopulations (villous and extravillous trophoblasts). To better understand differentiation and function of trophoblast components, we studied the distribution patterns of CKs in the placenta throughout pregnancy. A panel of well-defined monoclonal antibodies against different types of cytokeratins, vimentin, and fibrin, was used on frozen and paraffin sections. CK8, 18, and 19 were expressed in all the villous and extravillous trophoblastic subsets throughout pregnancy. In the first trimester, syncytiotrophoblasts were positive for CK7 and 13 along the basal membrane. As pregnancy progressed there was an increase in intensity of the reaction product and a more diffuse positive staining of CK7 in the cytoplasm of the syncytium, with evident positivity along the apical membrane. CK13 showed similar expression as CK7, but with less intense staining along the apical membrane and less prominent staining in the cytoplasm. Villous cytotrophoblasts were also positive for CK7 and CK13. CK17 was found related to cytotrophoblastic cells in contact with or next to fibrin deposits. Extravillous cytotrophoblasts in cell islands and cell columns were positive for CK13 only in the cell layers located proximal to the villous stroma, whereas the distal and more differentiated cells were negative. CK7 was positive in all epithelial cells of cell islands and columns, but the reaction product was not present in cells deeply migrated into the decidua. Amnion was negative for anti-CK13 antibodies in the first trimester but was positive at term. CK4 and CK16 were not found in the placenta. Our study shows for the first time that the different populations of human placental trophoblast express cytokeratins in developmental, differentiative, and functional specific patterns. These findings can be useful to distinguish and classify the various trophoblastic populations and provide a foundation for studying pathological aspects of the trophoblast.

Differentiation and proliferation patterns in human trophoblast revealed by c-erbB-2 oncogene product and EGF-R.

It is well known that growth factors and proto-oncogenes play a pivotal role in organogenesis as well as in tumor development. The human placenta is a rapidly growing organ which shares some aspects with malignant tumors. We have studied the expression of epidermal growth factor receptor (EGF-R) and the receptor encoded by the c-erbB-2 proto-oncogene in first- and third-trimester human placentas. We compared these expression patterns with that of the proliferation marker Ki-67. By immunohistochemistry, EGF-R was intensively expressed in the villous cytotrophoblast in the first trimester. The apical plasma membrane of the syncytium was weakly stained. In placental villi from the third trimester the reaction product for EGF-R was most intense in single villous cytotrophoblastic cells and along the apical plasma membrane of the syncytium, whereas the basal plasma membrane was much less stained. C-erbB-2 protein product was expressed in the first and third trimesters along the apical membrane of the syncytiotrophoblast. Concerning the extravillous trophoblast in cell islands and cell columns, EGF-R was expressed in the cells proximal to the villous stroma whereas the distal cells were c-erbB-2 positive. The Ki-67 antibody revealed the proliferative character of the villous cytotrophoblast and of the EGF-R-positive extravillous trophoblast. In contrast, most of the c-erbB-2-positive cells were Ki-67 negative. By in situ hybridization, c-erbB-2 transcripts were found in all types of villous and extravillous trophoblast, including those that did not express c-erbB-2 protein product. Our data indicate that EGF-R expression is strongly related to the proliferative trophoblast and, with advancing pregnancy, to the differentiated villous trophoblast.off contrast, expression of c-erB-2 protein product occurs only in more advanced stages of trophoblast differentiation, although transcripts of c-erbB-2 are found in both proliferative and differentiated trophoblast. In addition, the coexpression of EGF-R and c-erbB-2 protein product in the syncytiotrophoblast suggests their involvement in complex regulation of hormones and growth factors.

A three-dimensional study of the normal human placental villous core: II. Stromal architecture.

The three-dimensional architecture of the villous core of the chorionic villi has been studied in the human placenta throughout pregnancy by scanning electron microscopy, and compared with light microscopic and transmission electron microscopic, findings. Five different types of villi are recognizable: the mesenchymal villi are characterized by a network of tiny bundles of collagen fibrils, enmeshing mesenchymal cells, Hofbauer cells and vessels. The immature intermediate villi display a system of intercommunicating channels, parallel to the major axis of the villi. Within the channels, Hofbauer cells are present. The mature intermediate villi show narrow and short stromal channels, surrounded by a rather tight network of collagen fibrils. Large vessels and a highly compact network of collagen are typical of the stem villi. The terminal villi contain sinusoidally dilated capillaries and a small amount of connective tissue. These findings have numerous functional implications. They are particularly related both to the presence of the stromal channels which could facilitate the macrophagic and immunological task of the Hofbauer cells, and to the different types of villi, playing different roles in placental and fetal life, under normal and pathological conditions.

Branching patterns of human placental villous trees: perspectives of topological analysis.

Topological analysis was applied to investigate the branching pattern of three specimens obtained from early human placenta (6, 9, and 16 weeks p.m.) reconstructed on the basis of semi-thin sections. Centripetal Horton-Strahler and centrifugal branching order nomenclature was used for topological description of the analysed tree-like structures. Bifurcation ratio and vertex ratio were determined for all three cases and were found to be relatively constant. It was shown that branching pattern is closely related to the model of random segment branching that implicates a high level of asymmetry and a small level of space limitation for branching. The significance of this approach for the analysis of development of the villous tree, for the analysis of mesenchymal villous heterogeneity, and for the estimation of physiological parameters for fetoplacental exchange is discussed. We suggest that topological analysis can lead to a new quantitative classification of branching patterns of the human placental villous trees in normal and pathologic pregnancies.

Mitosis of the Hofbauer cell: possible implications for a fetal macrophage.

Human chorionic villi from placentae collected during the first half of pregnancy were examined by light microscopy and transmission electron microscopy. In serial semithin sections mitoses of Hofbauer cells, as well as those of other cellular components of the villous stroma, were generally easily identified. In some cases, when the Hofbauer cells showed very few vacuoles, thin sections were helpful in differentiating this cell type from the fixed stromal cells. Hofbauer cells in mitotic division were not uncommon, but were irregularly distributed. Mitosis of the Hofbauer cells could be a mechanism involved in maintaining the permanent presence in the chorionic villi of cell subpopulations with different origins and functions. Another explanation for the mitotic division of these cells could be that they are a self-renewing population independent of fetal monocytes which appear later in gestation in chorionic villi. In addition, mitosis of the Hofbauer cells could allow a rapid increase in their numbers when required by the local microenvironment.

Urokinase receptor is up-regulated in endothelial cells and macrophages associated with fibrinoid deposits in the human placenta.

Clearance of fibrin deposits within the human placenta is an ongoing process during normal placental development. Plasminogen is a circulating fibrinolytic protease zymogen activated in situ by plasminogen activators. We have previously reported that the receptor for urokinase plasminogen activator (uPAR) is expressed by cells either covering or enmeshed within the perivillous fibrinoid deposits. Whereas these cells seemed likely to be trophoblasts, a definitive identification was lacking, and this question is central to the understanding of the cellular mechanisms directing fibrinolysis in the placenta. In this study we have performed immunohistochemical co-localization studies and found that the uPAR-positive cells covering fibrinoid deposits are immunoreactive for CD31 and vWF, indicating that they are actually endothelial cells. In addition, we found that perivillous fibrinoid deposits not covered with uPAR-positive endothelial cells were covered with platelets identified by integrin alpha(IIb)beta(3)-immunoreactivity. Also surprisingly, the uPAR-positive cells enmeshed within fibrinoid deposits express a cell specific marker indicating that they are macrophages. Both uPAR-positive cell populations also express uPA immunoreactivity. Taken together, the data suggest that both fibrinoid-covering endothelial cells and fibrinoid-enmeshed macrophages can participate in the clearance process of perivillous fibrinoid deposits formed in the human placenta.

Immunohistochemical identification of the receptor for urokinase plasminogen activator associated with fibrin deposition in normal and ectopic human placenta.

The receptor for urokinase plasminogen activator (uPAR) is a key molecule in cell surface-directed plasminogen activation. uPAR binds urokinase plasminogen activator (uPA) and thereby focuses plasminogen activation on the cell surface. Plasmin dissolves fibrin deposits and facilitates cell migration during tissue repair processes by degrading the extracellular matrix. During human implantation and placental development, plasmin is considered important for both trophoblast migration/invasion and for fibrin surveillance. This study examined the expression of uPAR in normal and ectopic human placentae by immunohistochemistry. In first and third trimester normal placentae as well as in tubal ectopic placental tissues, a high uPAR expression was seen in the trophoblast associated with deposits of fibrin-type fibrinoid. Extravillous trophoblast of the basal plate, of the cell islands, and of the cell columns was also positive for uPAR in the first trimester whereas at term the expression of the protein was decreased. Moreover, uPAR immunostaining was observed in decidual cells throughout normal gestation and in endometrial tissues of patients with ectopic pregnancies. These findings suggest that uPAR participates in placental development and in trophoblast invasion particularly in the first trimester of pregnancy and that uPAR is involved in repair mechanisms of the trophoblast and fibrin surveillance.

The development of the human placental villous tree.

The present investigation was undertaken in order to achieve a better understanding of the dynamics of placental villous differentiation. Villous trees from human placentas from different stages of pregnancy (first trimester to full term) were isolated and studied by light microscopy and scanning electron microscopy. For light microscopy the trees were serially sectioned and two-dimensionally reconstructed. For scanning electron microscopy complete villous trees or freeze-cracked villi were studied. The most important finding was that the mesenchymal villi are continuously newly formed out of the trophoblastic sprouts throughout pregnancy. Because of this they exist in all stages of pregnancy and have to be considered the basis for growth and differentiation of the villous trees. In the first two trimesters they are the forerunners of the immature intermediate villi, whereas in the last trimester the mesenchymal villi are transformed into mature intermediate villi. The immature intermediate villi formed during the first two trimesters are developmental steps towards the stem villi. On the other hand, the mature intermediate villi, which only are developed during the last trimester, produce numerous terminal villi. The latter are not active outgrowths caused by proliferation of the trophoblast, but rather passive protrusions induced by capillary coiling due to excessive longitudinal growth of the fetal capillaries within the mature intermediate villi.

S-100 protein localization in human adult dental pulp.

The pulp of third molar teeth was examined by immunofluorescence and immunoperoxidase using a specific antibody to detect S-100 protein-labelled cells. There was a strong positive reaction in macrophages and in Schwann cells ensheathing axons. The other cells within the pulp were negative.

Fibronectins and basal lamina molecules expression in human subcutaneous white adipose tissue.

Adipose tissue is a type of connective tissue whose extracellular matrix (ECM) components are poorly characterized in vivo. Several in vitro studies have suggested a regulatory role for the ECM molecules during adipocyte differentiation. Since no data are available concerning the in vivo expression of the main ECM components such as fibronectin, collagen IV, laminin and heparan sulfate proteoglycan in the adipose tissue, we investigated the presence of these molecules by immunohistochemistry. We show that fibronectin isoforms are not expressed in fully differentiated subcutaneous adipocytes whereas collagen IV, laminin and heparan sulfate are detectable around single adipocytes, i.e. in the region corresponding to the basement membrane. These data are supported by previous in vitro studies showing a strong decrease of fibronectin synthesis during adipocyte development whereas basement membrane molecules seem to increase during adipocyte differentiation.

Hyaluronate and CD44 expression patterns in the human placenta throughout pregnancy.

Hyaluronan (HA) and CD44 are involved in several processes such as cell migration and differentiation. In the present study, we examined the expression and distribution of both hyaluronan and its cell surface receptor (CD44) in the human placenta, which is a rapidly growing and differentiating organ that plays a fundamental role in fetal life. Hyaluronan was detected by a specific biotinylated binding probe, termed b-PG. In the first half of gestation, HA was strongly expressed in the stroma of the mesenchymal villi which have been previously identified as responsible for the growth and differentation of the villous trees. The other villous types showed an intense staining only in the fetal vessel walls and in the connective tissue closely underlying the trophoblastic cover. In addition, hyaluronan positive staining was also apparent in a restricted rim of villous stroma directly apposed to extravillous cytotrophoblastic cell islands and cell columns. In full term placentas, all villi expressed HA in their stromal tissue with a more homogenous staining than in the first half of gestation. In contrast to hyaluronan, in the first trimester CD44 was restricted to some of the Hofbauer cells which may be able to internalize hyaluronan, thus playing a significant role in its removal in early pregnancy. CD44 was primarily expressed starting from the 16th week of gestation. At the end of pregnancy it was expressed in the various villous types, especially in stem villi. Moreover, the plasma membrane of some extravillous cytotrophoblastic cells in the basal plate and the large majority of the decidual cells showed a positive immunostaining for this receptor. Taken together, these data suggest that HA is strongly involved in early villous morphogenesis, whereas CD44 seem to be play an important role in tissue remodelling later in gestation.

IL-1ß and TGF-ß weaken the placental barrier through destruction of tight junctions: an in vivo and in vitro study.

INTRODUCTION: Chorioamnionitis is a gestational pathological condition characterized by acute inflammation of the amniochorionic membranes and placentas leading to high concentrations of IL-1ß, Il-6, Il-8 and TGF-ß in the amniotic fluid. In normal conditions, the permeability of foeto-maternal barrier is due to the assembly and maintenance of different cellular junctional domains. METHODS: In the present study, first we aimed to evaluate the protein expression (by immunohistochemistry and western blotting) and mRNA (by real time PCR) levels of the molecular components of tight junctions (Zonula occludens-1 and occludin), and of adherent junctions (VE-cadherin and ß-catenin) in placentas from chorioamnionitis compared to that in normal pregnancies. RESULTS: Western blotting results showed a significant down-regulation of occludin in placentas affected with chorioamnionitis. No differences were detected for the other proteins analysed. We evaluated whether occludin expression was regulated by IL-1ß, IL-6, IL-8 and TGF-ß by means of in vitro studies using HUVEC cultures and demonstrated a key role of IL-1ß and TGF-ß in the disappearance of occludin at cellular border. CONCLUSIONS: We conclude by suggesting a pivotal role of these two cytokines in facilitating intra-placental infection via para-cellular way due to the disassembly of tight junctions at trophoblastic and endothelial cells in placental tissues.