Self-regulation of the endothelin receptor system in choriocarcinoma cells.

The human trophoblast secretes endothelin-1 (ET-1) and expresses ET receptors. The present study tested whether the transformed BeWo, JAR and JEG-3 choriocarcinoma cells: (1) secrete endothelin-1 (ET-1); (2) express both ET-A and ET-B receptor subtypes; and (3) have the potential to allow for autologous regulation of ET-receptor proteins. The cells were cultured for 24/48 h with or without 10% FCS and, in experiments on receptor regulation, with ET-1 (5-20 nM and 10 microM). ET-1 secretion was measured by RIA and receptor levels by immunoblotting. All cell types secreted ET-1 albeit at different levels and sensitivity to FCS. All cell lines expressed both ET-A (JEG-3>BeWo=JAR) and ET-B (JEG-3=JAR>BeWo) receptor subtypes, which could be up- and downregulated depending on ET-1 concentration, culture time and FCS presence. It is concluded that BeWo, JAR and JEG-3 choriocarcinoma cells secrete ET-1 and express both ET-A and ET-B receptor subtypes. The receptor levels can be regulated by ET-1. This provides the molecular basis for an autocrine system with the potential of autologous regulation of yet unidentified ET-1-induced functions.

Endothelin A and B receptors change their expression levels during development of human placental villi.

Endothelin receptors have recently been found in non-vascular tissues including the human placenta. The present study investigated developmental changes in location and expression levels of endothelin A and B receptors (ETA-R, ETB-R) in human placentae and isolated trophoblast by comparing first and third trimester tissues. In the first trimester all cells and tissues were immunolabelled for ETA-R and ETB-R, whereas in third trimester placentae the syncytiotrophoblast (ETA-R, ETB-R) and macrophages (ETA-R) were unstained. Immunoblotting for both receptors revealed up to three bands at 33-35, 50 and 75 kDa, respectively, which were differentially present in the first and third trimester. Pre-adsorption of antibodies with oligopeptides used for antigen-generation weakened the immunoreactions. ETA-R protein levels decreased (P< 0.05) in total villous tissue and isolated trophoblast, whereas ETB-R was unchanged. ETB-R transcripts (RT-PCR) prevailed in both stages and tissues, but in contrast to the protein levels its preponderance decreased from first trimester to term in villous tissue (P< 0.01), because of a four to five-fold increase in ETA-R and only a two-fold (P< 0.05) increase in ETB-R mRNA levels (P< 0.01). We conclude that ET receptor location, intracellular processing and expression levels in human villous tissue change between the first and third trimester. This may reflect changing functions of ET-1 during placental development.

Pre-eclampsia and gestational age differently alter binding of endothelin-1 to placental and trophoblast membrane preparations.

The aim of the study was to compare the binding of endothelin-1 (ET-1) to membranes from placental tissue and trophoblast cells in normal and pre-eclamptic pregnancies. Plasma membranes from placental tissue and trophoblastic cells were prepared from 15 control and 18 pre-eclamptic pregnancies at either preterm (weeks 31-36) or term (weeks 37-40). ET-1 binding to tissue membranes was measured by a radioreceptor assay. In addition, binding of 56 nmol/l [125I]ET-1 to plasma membranes of trophoblastic cells was determined. In pre-eclampsia, placental membranes bound less (P < 0.01) ET-1 owing to fewer (P < 0.01) receptors at preterm than in the corresponding preterm controls. In contrast, binding of [125I]ET-1 to plasma membranes of trophoblast cells was higher (P < 0.01) in pre-eclampsia at both gestational stages than in the controls. Incubation of trophoblast cells with hydralazine reduced binding by 70%. We conclude that pre-eclampsia is associated with changes in the binding of ET-1 to its placental receptors. Moreover, the data suggest that pre-eclampsia affects non-trophoblast cells in the opposite manner to the trophoblast.

Endothelin-1 stimulates the proliferation and invasion of first trimester trophoblastic cells in vitro--a possible role in the etiology of pre-eclampsia?

BACKGROUND: Endothelin-1 is a potent mitogen and stimulates cell chemokinesis, but these properties have not been investigated in the placenta. Trophoblastic cells from pre-eclamptic pregnancies have a reduced invasive potency and secrete less endothelin-1 than those from normal pregnancies. The present study tested the hypothesis that endothelin-1 affects trophoblast proliferation and invasion. METHODS: Trophoblastic cells were isolated from 37 placentae of normal human pregnancies at week 12 by dispastrypsin digestion and subsequently immunopurified to remove nontrophoblastic components. The effects of 5, 10, and 20 nmol/mL endothelin-1 on proliferation and invasion were determined after 24 and 72 hours, respectively, by fluorescence-activated pulse cytometry (FACS) analysis, by measuring DNA synthesis using two different methods and by a Matrigel invasion assay. RESULTS: All cell preparations uniformly responded to 10 nmol/mL by increased proliferation, owing to a greater proportion of cells in the S-phase of their cell cycle, and invasion. The effects were more pronounced after 24 hours than after 72 hours, by which time cell viability, as measured by the secretion of human chorionic gonadotropin (hCG-beta), had deteriorated. The nonselective receptor antagonist PD 142893 inhibited both endothelin-1 effects. CONCLUSION: Endothelin-1 is a mitogenic stimulus for first trimester trophoblastic cells in vitro. The stimulation of cellular invasion represents a novel effect of endothelin-1. We suggest the implication of endothelin-1 in proliferation and invasion of trophoblast and tumour cells and hypothesize a possible role in the etiology of pre-eclampsia.

Effects of parathyroidectomy on tissue calcium, phosphorus, magnesium, and copper concentrations in aluminum-loaded uremic rats.

Rats were subjected to a two-stage 5/6 nephrectomy and treated with Al for 2 and 4 wk with a cumulative dose of 4.2 and 8.4 mg of Al, respectively. Other animals were parathyrectomized (PTx) and loaded with 8.4 mg of Al for 4 wk. Total Al, Ca, P, Mg, and Cu contents were analyzed in the liver, kidney, and bone by inductively coupled plasma atomic emission spectrometry (ICP-AES). The results showed that Al given to growing uremic rats significantly increased the content of Al in the liver, kidney, and bone. Moreover, Al treatment increased the liver and kidney Ca levels and decreased the Ca and P values in bone. Previous parathyroidectomy significantly reduced Al accumulation within organs and changes in the Ca and P levels in the bone, liver, and kidney. The result was not influenced by different degrees of renal failure.

[Secretion of endothelin-1 by trophoblast cells and binding of endothelin-1 to trophoblast membranes in healthy and pre-eclampsia pregnancies]. / Sekretion von Endothelin-1 aus Trophoblastzellen und Bindung von Endothelin-1 an Trophoblastmembranen bei gesunden und präeklamptischen Schwangerschaften.

OBJECTIVES: The hypothesis was tested that the synthesis or binding of endothelin-1 (ET-1) by the trophoblast are altered in preeclampsia. METHODS: Trophoblast cells (TC) were isolated from 15 normal (C) und 18 pre-eclamptic pregnancies (P). Binding of [125I]ET-1 to plasma membranes of TC was determined after 48 h in culture. Concentration of ET-1 in culture media were measured after each 24 h over 5 day culture period. RESULTS: Specific binding was 150% (week 37-40) - 400% (week 31-36) higher (P < 0.01) in P. In contrast, the release of ET-1 was 60% (week 31-36) - 71% (week 37-40) lower (P < 0.01) in P than in healthy controls. CONCLUSIONS: Pre-eclampsia is associated with a lower release and increased binding of ET-1 by trophoblast cells.

[Fractures with loss of substance of the middle and upper third of the face: nosographic classification, surgical indications and the prevention of meningeal infections with the new antibiotic, cefuroxime]. / Le fratture con perdita di sostanza del terzo medio e superiore della faccia: inquadramento nosografico, indicazioni chirurgiche, profilassi dell infezioni meningee con il nuova antibiotico cefuroxim.

Reference is made to considerable personal experience acquired in the treatment of craniofacial fractures, particularly those affecting the upper third of the face. An account is given of the anatomical and surgical features of the naso-ethmoid-frontal and temporo-orbito-sphenoid areas--now recognised as within the domain of maxillofacial surgery--and their symptomatologies. Particular attention is directed to the question of liquorrhoea and the possibility of ascendent infections of the meninges. Reduction is favoured with respect to the former, since forward and upward repositioning often leads to the regression of ethmoidal liquorrhoea. Exclusion of the frontal sinus is regarded as a "categorical imperative" as far as meningeal infection is concerned, and support is also given for its prevention with antibiotics. A personal preference is expressed for cefuroxim, a new antibiotic with particular potency and marked diffusibility within the cerebrospinal fluid.

[Aluminum poisoning]. / Otrovanje aluminijem.

Aluminum intoxication is common in patients with chronic renal failure because of absorption of aluminum during dialysis from aluminum-containing dyalysate water and ingestion of phosphate binders containing aluminum. Aluminum accumulation in the body is followed by bone disease, encephalopathy and anemia. Bone diseases can be recorded in 44% of the patients treated with long-term dialysis. Two early histologic types of retarded bone turnover can be seen, i.e. osteomalacia and aplastic bone disease. In dialyzed patients, osteomalacia is usually followed by low PTH level in human serum. On the contrary, studies on uremic rats have shown that previous parathyroidectomy can prevent aluminum intoxication, because hyperparathyroidism in an early phase of chronic renal failure increases aluminum absorption from the gut and its accumulation in the body. As the pathogenesis of aluminum-induced alterations is unclear, the prevention of bone disease should be provided through lowering the aluminum intake in dialyzed patients. Bone biopsy is unavoidable for the early detection and diagnosis of the disease. Promising results in the treatment of aluminum intoxication have been obtained using deferoxamine, a chelating agent.

Altered release of endothelin-1,2 and thromboxane B2 from trophoblastic cells in pre-eclampsia.

The aim of the study was to investigate whether pre-eclampsia is associated with an altered release of vasoactive substances from trophoblastic cells in vitro. Trophoblastic cells from 15 uncomplicated control pregnancies and 18 pre-eclamptic pregnancies at preterm (weeks 31-36; n = 12) and term (weeks 37-40; n = 21) were cultured for 5 days. The concentrations of angiotensin II (AII), endothelin-1,2 (ET-1,2), thromboxane B2 (TXB2), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and leukotriene B4 (LTB4) were measured daily in culture media for 5 days by radioimmunoassay. In pre-eclampsia, concentrations of ET-1,2 were decreased (P < 0.01) at both preterm and term, TXB2 concentrations were increased (P < 0.05) only at preterm and the TXB2-6-keto-PGF1 alpha ratio was increased at both preterm and term (P < 0.01) as compared with the controls. Concentrations of AII, 6-keto-PGF1 alpha and LTB4 were similar to the controls. The data suggest that pre-eclampsia is associated with a decreased release of ET-1 and an increased release of TXB2 from trophoblastic cells in vitro.

Drug actions in preeclampsia: aspirin, but not magnesium chloride or dihydralazine, differentially inhibits cultured human trophoblast release of thromboxane and prostacyclin without affecting angiotensin II, endothelin-1, or leukotriene B4 secretion.

OBJECTIVE: We hypothesized that aspirin Mg++, and dihydralazine affect the release of vasoactive agents from cultured human placental trophoblast. STUDY DESIGN: Cytotrophoblasts isolated from placentas of preterm or term deliveries of 14 healthy control women and 15 preeclamptic women were cultured in Dulbecco's modified Eagle's medium for 5 days in the presence or absence of either 0.1 mmol/L aspirin, 3 mmol/L magnesium chloride, or 136 ng/ ml dihydralazine. Vasoactive substances were quantitated by radioimmunoassay with mean +/- SEM percentage of untreated cells (= 100%) compared by the Mann-Whitney U test and analysis of variance. RESULTS: Aspirin inhibited (p < 0.01) both thromboxane and prostacyclin on days 1 and 2 in culture but not on days 3 to 5 unless the Dulbecco's modified Eagle's medium was supplemented with arachidonic acid. Aspirin inhibition was greater (p < 0.01) for thromboxane in cells cultured 24 hours after preeclamptic pregnancy (preterm 29.9% +/- 6.8%, term 20.1% +/- 5.9%) compared with normal controls (preterm 66.3% +/- 10.6%, term 68.9% +/- 11.6%). Aspirin reduced (p < 0.01) the ratio of thromboxane to prostacyclin in media of cells from preeclampsia (untreated 27.8 +/- 7.2, aspirin 13.3 +/- 4.4), but aspirin had no effect on this ratio in cultures from control normal pregnancies (untreated 6.8 +/- 2.9, aspirin 4.8 +/- 1.1). Neither magnesium chloride nor dihydralazine affected trophoblast prostanoid production, and no drug altered the media levels of angiotensin II, endothelin-1, or leukotriene B4. CONCLUSION: Aspirin selectively inhibits trophoblast prostanoid production. This inhibition depends on the availability of arachidonic acid and the presence or absence of preeclampsia. Magnesium and dihydralazine effects in pregnancy are not related to altered release of trophoblast vasoactive compounds.

Paracrine regulation of distinct trophoblast functions in vitro by placental macrophages.

In view of the accumulating evidence for paracrine mechanisms regulating trophoblast function, we tested the hypothesis that placental macrophages affect trophoblast activity in a paracrine fashion. Trophoblast was isolated from 17 term placentas (-IP). One aliquot of cells was further immunopurified (+IP) using an HLA class I antibody. This increased the proportion of trophoblast (+IP >97%; -IP approximately 70%) as identified by rigorous immunocytochemistry. Most (approximately 70%) non-trophoblast cells in -IP were macrophages. The cells were cultured for 5 days with a daily medium change. In addition, +IP cells from seven placentas were cultured with lipopolysaccharide (LPS)-stimulated or -unstimulated macrophage-conditioned media. The concentrations of lactate, trophoblast-specific hormones, human chorionic gonadotropin-beta (hCG-beta) and human placental lactogen (hPL), of several prostanoids and of endothelin-1 and angiotensin II were determined in the culture media. The accumulated amounts of substances released into the culture media, corrected for the greater proportion of trophoblast in +IP cultures, were on average two- to threefold higher (hCG-beta: 18-fold) in +IP than in -IP, with the exception of endothelin-1,2 (no change), angiotensin II (-70%) and 6-keto-prostaglandin-F1alpha (-40%). [3H]leucine incorporation into the trichloroacetic acid (TCA)-precipitable pool measured on day 5 was twofold higher in +IP than in -IP. Addition of conditioned media reverted these changes. The data demonstrate that placental macrophages in culture affect trophoblast biosynthetic activity in a paracrine fashion. We conclude that macrophages are important regulators of trophoblast activity.

The influence of early parathyroidectomy on aluminum-induced rickets in growing uremic rats.

Rats were subjected to a two-stage 5/6 nephrectomy and treated with aluminum for 2 and 4 weeks with a cumulative dose of 4.2 and 8.4 mg of aluminum, respectively. Other animals were parathyroidectomized and loaded with 8.4 mg of aluminum for 4 weeks. Histomorphometry and electron microscopy (tibiae), aluminum tissue (bone, kidney, liver) determination, serum (Ca, Mg, Zn, P, urea, creatinine, alkaline phosphatase, 1,25(OH)2D3, PTH) and urine (creatinine, A1) revealed that: (a) a dose of 8.4 mg aluminum was sufficient to induce rickets within 4 weeks of treatment and was associated with decreased serum calcitriol values and high aluminum accumulation within organs (electron-dense material was found in osteoblasts only); (b) previous parathyroidectomy prevented the occurrence of any aluminum-induced alteration of bone. It was associated with higher calcitriol and phosphorus values than in corresponding non-parathyroidectomized rats and significantly reduced aluminum accumulation within organs. The results was influenced neither by a drop in serum calcium values nor by different degrees of renal failure. We suggest that aluminum-induced rickets in growing uremic rats is prevented or delayed when previous parathyroidectomy has been performed.

Antithrombin III in fetuses and newborn children.

Antithrombin III (AT III) was measured in plasma of healthy fetuses between 20-29 weeks of gestation, in plasma of suspected growth retarded or malformed fetuses between 30-39 weeks of gestation, in healthy newborn infants after delivery, and in healthy infants during the first year of life. Measurements were performed with an AT III assay (Orion Diagnostica, Espoo, Finland Turbox) on nephalometer Turbox. The results were expressed as a percentage of the mean adult value (300 mg/l) and statistically analysed with the non-parametric Kruskal-Walles test. AT III levels in fetuses were low but increasing. They continued to increase after birth (F = 34.53 p < 0.001) and reached adult values in the age between the tenth and twelfth month of life.

Differential regulation of endothelin secretion and endothelin receptor mRNA levels in JAR, JEG-3, and BeWo choriocarcinoma cell lines and in human trophoblasts, their nonmalignant counterpart.

Endothelin (ET) secretion and expression of both ET-A and ET-B receptor subtypes have been found in a number of primary cancers. The present study tested (1) whether choriocarcinoma cells and their nonmalignant counterpart, the trophoblast, secrete ET-1 and express ET-A and ET-B receptors; (2) whether ET-1 secretion and receptor mRNA levels are regulated by the same factors in nonvascular tissues as in vascular tissues; and (3) whether such regulation is similar in malignant and nonmalignant cells. All cells secreted ET-1 in similar amounts (approximately 0.8 fmol/10(6) cells per 24 h) and secretion was unaffected by culture and treatment. Whereas ET-B accounted for almost all (>98%) ET receptor transcripts in the choriocarcinoma cells, the trophoblasts expressed about 20% ET-A receptor mRNA. During control cultures, ET-B mRNA levels rose in choriocarcinoma, with the greatest relative increase (6-fold; P < 0.05 vs 0 h) in BeWo, whereas in trophoblasts, ET-A mRNA transiently changed after 24 and 48 h. Treatment with dexamethasone and glucose did not alter the mRNA levels in all cells. Insulin induced changes (P < 0.05) in ET-B mRNA levels in BeWo (+90 and +60% after 24 and 48 h, respectively) and JEG-3 (-70%), but not in JAR and trophoblast cells. We conclude that malignant transformation affects the responsiveness of the endothelin receptor system to external stimuli and that the regulation of the endothelin system differs in vascular and nonvascular tissues.

Hyperglycaemia in vitro alters the proliferation and mitochondrial activity of the choriocarcinoma cell lines BeWo, JAR and JEG-3 as models for human first-trimester trophoblast.

AIMS/HYPOTHESIS: Early intrauterine growth delay in diabetes could be caused by a reduced growth of the placenta. Our study investigates whether hyperglycaemia in vitro reduces trophoblast proliferation. METHODS: First-trimester trophoblast cell models (BeWo, JAR and JEG-3 choriocarcinoma cells) were cultured for 24 and 48 h with 5.5 mmol/l D-glucose, 25 mmol/1 D-glucose (hyperglycaemia) and with an osmotic control. Cell number, total protein and nucleic acid content and mitochondrial activity (tetrazolium salt assay) were measured, the cell cycle analysed (FACS, cyclin B1 levels) and apoptosis (Annexin-V) measured. RESULTS: In BeWo cells hyperglycaemia reduced cell number, protein, nucleic acid and cyclin B1 levels. The reduced G2/M and increased G0/G1 population after 24 h reflects growth arrest at G0/G1. In JAR cells after 24 h the population was arrested in G0/G1, whereas after 48 h the G0/G1 block was abrogated and the cells were arrested at G2/M. The net effect was an unchanged cell number. In JEG-3 cells hyperglycaemia resulted in fewer cells after 24 h but not after 48 h indicating some adaptation. Mitochondrial activity was either stimulated (BeWo) or reduced (JAR, JEG-3) under hyperglycaemia. Some of these effects were also induced by hyperosmolarity alone. CONCLUSION/INTERPRETATION: Hyperglycaemia has the potential to inhibit the proliferation of first-trimester trophoblast cell models. The mechanisms leading to growth arrest and to changes in mitochondrial activity are complex and depend on differentiation. We hypothesise a hyperglycaemia-induced impairment of placental growth in the first trimester of a poorly controlled diabetic pregnancy.