Hypoxia up-regulated angiogenin and down-regulated vascular cell adhesion molecule-1 expression and secretion in human placental trophoblasts.

OBJECTIVE: Many processes that are involved in cellular invasion, including blastocyst implantation, placental development, and rapidly growing tumors, occur in reduced oxygen environments. It has been surmised that oxygen tension could regulate the cytotrophoblast ability to differentiate and, as a consequence, to express proteins that are critical for placentation. The objective of the current investigation was therefore to test the hypothesis that placental tissues and trophoblast cells in culture, under low oxygen tension, release angiogenic factors that could affect vascular behavior and invasive potential, thus providing a link between abnormal placentation and maternal vascular abnormality. METHODS: Functionally active term placental explant culture and trophoblast cultures were used to demonstrate the secretion profiles of angiogenin and vascular cell adhesion molecule-1 (VCAM-1), and the real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique was employed to demonstrate the mRNA expression under both normoxic and hypoxic conditions. RESULTS: A significant increase in the secretion (P <.01) and mRNA expression (P <.01) of angiogenin and a significant decrease in the secretion (P <.04) and mRNA expression (P <.03) of VCAM-1 from both term placental explants and trophoblast cultures subjected to hypoxia in vitro were observed. CONCLUSION: Because the primary defect in uteroplacental insufficiency is placental maldevelopment probably associated with hypoxia in situ, this study provides molecular evidence to indicate that the differential expression and secretion of angiogenic factors may play an important role in these pathologic conditions.

Resistance to Fas-mediated cell death in BeWo and NJG choriocarcinoma cell lines: implications in immune privilege.

OBJECTIVE: An immune privileged site occurs when the allogenic tissue grafts have the propensity for prolonged survival in the host tissue. In this context, the survival and proliferation of malignant trophoblasts in the gravid uterus are currently unclear. In a previous study, we documented that Fas and FasL are coexpressed in choriocarcinoma [Gynecol. Oncol. (2003)]. This study was conducted to examine the role of the Fas/FasL pathway in immune privilege of BeWo and NJG choriocarcinoma cells in culture. METHODS: The ability of anti-Fas mAb (CH-11) to sensitize choriocarcinoma cell lines to Fas-mediated cytotoxicity was assessed by MTT assays. Coculture experiments with Fas-sensitive Jurkat cells were used to demonstrate functional FasL from choriocarcinoma. RT-PCR was used to assess the expression of cFLIP. RESULTS: The mean cell viability of BeWo and NJG cells declined to about 58 and 63% compared to controls after 72 h of culture in the presence of anti-Fas mAb (CH-11) while the Fas-sensitive Jurkat cells showed viability of only 10%. This resistance to Fas-mediated apoptosis in choriocarcinoma cells is reversed in the presence of cycloheximide (0.5 micro g/ml) which further decreased the viability to 36 and 32%, respectively, at a dose of 300 ng/ml (P < 0.05). The observed resistance to Fas-mediated apoptosis therefore could be attributed to the short-lived endogenous inhibitor, cFLIP as demonstrated by the RT-PCR technique. In coculture experiments, FasL from choriocarcinoma cells induced apoptosis in the Fas-sensitive Jurkat cells, thereby indicating the capacity to evade immune attack. CONCLUSIONS: Decreased sensitivity to Fas-mediated apoptosis and counterattacking the lymphocytes may impart immune privilege in these malignant trophoblasts for prolonged survival in the host.

Co-expression of Fas (APO-1, CD95)/Fas ligand by BeWo and NJG choriocarcinoma cell lines.

OBJECTIVE: Fas (CD95) is a transmembrane protein of the tumor necrosis factor receptor superfamily that induces apoptosis in susceptible cells on crosslinking by its ligand (FasL). The Fas loss of function and concurrent expression of its ligand (FasL) have been associated with malignant phenotype. In this study, we sought to investigate the hitherto undescribed expression of Fas and FasL on the immortalized human choriocarcinoma cell lines BeWo and NJG. METHODS: Receptor and ligand expression was demonstrated using specific antibodies and multiple techniques including immunocytochemistry, confocal immunofluorescence microscopy, flow cytometry, immunoblots, and reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Data from this study indicate that human choriocarcinoma cell subtypes co-express both Fas and FasL. A specific cytoplasmic and membranous pattern of immunoreactivity was noted that was further confirmed at mRNA transcripts by RT-PCR. In addition, we provide evidence using flow cytometry that the Fas receptors are downregulated. The mean fluorescence intensities for NJG and BeWo were 1.47 +/- 0.5 and 1.59 +/- 0.4, while that for Fas-positive Jurkat cells was 25.6 +/- 3.1. CONCLUSIONS: To our knowledge, this is the first report on the identification and constitutive co-expression of Fas and FasL in BeWo and NJG choriocarcinoma cells. Choriocarcinoma cells evade immune attack by downregulating the Fas receptor and by killing lymphocytes through expression of FasL. Taken together, our investigations suggest that the Fas/FasL system may represent a mechanism by which malignant trophoblasts become resistant to apoptosis, escape immune surveillance, and metastasize.

Expression and secretion of the vascular cell adhesion molecule-1 in human placenta and its decrease in fetal growth restriction.

OBJECTIVE: The vascular cell adhesion molecule-1 (VCAM-1) is a member of the immunoglobulin gene superfamily and is expressed principally on endothelial cells. The present study was undertaken to compare the expression and secretion of VCAM-1 from normal pregnancies and those complicated by fetal growth restriction (FGR). METHODS: Placentas from first-trimester (FT) (n = 17), normal term (n = 19), and FGR (n = 16) patients were collected immediately after elective cesarean delivery. VCAM-1 mRNA expression profiles by reverse transcriptase polymerase chain reaction, protein levels by enzyme-linked immunosorbent assay using explant culture in vitro, and its cellular localization by confocal microscopy were compared between FGR and normal placentas. RESULTS: Functionally active placental explants were used to detect immunoreactive VCAM-1 in conditioned media of all the samples from the three groups. The mean levels of VCAM-1 produced by FT villi were found to be 1.9-, 1.7-, and 1.5-fold higher (P <.02) than term villi at 24, 48, and 72 hours of culture, respectively. Conversely, the respective mean levels of VCAM-1 produced by FGR placental villi were 2.3-, 2.5-, and 2.0-fold lower than levels of the normal term placental villi (P <.05). The secretion profiles of VCAM-1 from FT, term, and FGR villi correlated well with the mRNA levels; the amount secreted in FT villi was twice that of term villi. Moreover, mRNA transcripts from FGR pregnancies showed significantly decreased expression, in conformity with explant results. CONCLUSIONS: The presence of VCAM-1 in placental villi and down-regulation of its production at term indicate that VCAM-1 production is specific to developmental stage. The decreased VCAM-1 expression in FGR pregnancy could be attributed to the uteroplacental deficiency that is characteristic of this condition.

Over-expression and secretion of angiogenin in intrauterine growth retardation placenta.

Human angiogenin is a potent inducer of neovascularization. There is a strong evidence to suggest that it might be involved in morphological and angiogenic changes in the placenta, that are necessary for a successful fetal outcome during pregnancy. However, its precise role in the pathogenesis of abnormal pregnancies is yet unknown. Intrauterine growth retardation (IUGR), an abnormal pregnancy is not a specific disease entity per se, but rather a manifestation of many possible fetal and maternal disorders. In this study, we demonstrated, for the first time, that placental explants in vitro secrete significantly elevated levels of angiogenin in placental tissues from patients with IUGR. We also observed enhanced mRNA expression in placenta from these patients. In addition, using the immunohistochemical methods, we observed identical staining of angiogenin to villous syncytiotrophobalst and fetal endothelial cells in both IUGR and normal placenta. Functionally active placental explants were used to detect immunoreactive angiogenin in conditioned media of all the samples from IUGR placenta and normal term group. The mean levels of angiogenin secreted by IUGR placenta were 1.4-, 1.6-, and 1.3-fold higher (P < 0.01) than normal term samples at 24, 48, and 72 hr of culture, respectively. Expression profiles of angiogenin from term and IUGR cases are in agreement with its mRNA levels and immunoblot analysis. In conclusion, the significant elevated levels of angiogenin in IUGR placenta may provide a molecular mechanism for the abnormal placental development.

Expression and localization of angiogenin in placenta: enhanced levels at term over first trimester villi.

Human angiogenin, a 14-kDa non-glycosylated polypeptide with both angiogenic and ribonucleolytic activities, is implicated in angiogenesis, a complex process of proliferation and formation of new capillary blood vessels from existing blood vessels. Placental growth requires extensive angiogenesis, which develops its vascular structure in both fetal chorionic villi and maternal deciduas. In this study, we investigated the expression profiles of angiogenin in placental villi from early and late gestation at both mRNA and protein levels using explant cultures in vitro followed by RT-PCR, immunoblot, and immunohistochemical analyses. From functionally active placental explants, angiogenin was detected in conditioned media of all the samples from first trimester and term group. The mean levels of angiogenin produced by term villi were found to be 2.6-, 2.1-, and 2.2-fold higher (P < 0.01) than first trimester villi at 24, 48, and 72 hr of culture, respectively. Expression profiles of angiogenin from term and first trimester villi seem to agree with its mRNA levels and immunoblot analysis; the expression in term villi was twice that in first trimester villi. The presence of angiogenin in placental villi and upregulation of its production towards term indicate that angiogenin production by the placenta is specific to the developmental stage. In conclusion, the observed changes in the localization and mRNA expression of angiogenin during placental development raise the possibility that it is involved in morphological and angiogenic changes in this endocrine organ vital to the successful fetal outcome during pregnancy.

Evidence for the biosynthesis of DHEA from cholesterol by first-trimester human placental tissue: source of androgens.

With a view to establishing whether first-trimester human placentas possess the ability to synthesize DHEA from cholesterol, homogenates of this tissue obtained from two groups of women undergoing elective termination of normally progressing pregnancy between 10 - 12 weeks gestation (n = 5, age 23 - 29 years and n = 5, age 21 - 27 years) were incubated separately with [26-(14)C]cholesterol for the generation of [14C]isocaproic acid + pregnenolone and [7n-3H]pregnenolone for the biosynthesis of [3H]DHEA. Controls consisted of homogenates heated in a boiling water bath for 10 min. Using the reverse-isotope dilution analysis, desmolase efficiency expressed as mean specific activity of [14C]isocaproic acid varied from 282 to 725 dpm/mmol, while that of 17 alpha-hydroxylase and steroid C-17,20-lyase, catalyzed conversion of [7n-3H]pregnenolone to [3H]DHEA varied from 3498 to 26 258 dpm/mmol. The corresponding efficiencies of enzymicconversion varied between 5.8 x 10( -2) and 1.5 x 10( -1) % for [14C]isocaproic acid, but between 5.5 x 10( -2) and 4.1 x 10( -1) % for [3H]DHEA. No such metabolite was evident in the controls of heat-denatured homogenates. These are the first study results to demonstrate that early placentas are capable of converting cholesterol to pregnenolone to DHEA, contrary to the widely held concept of DHEA production by fetal and maternal adrenal glands. This finding has important physiological implications and could provide a new dimension to the concept of fetoplacental steroidogenesis.

Enhanced dehydroepiandrosterone synthesis by amnion compared to chorion: a comparative study using the reverse-isotope dilution technique.

With a view to establishing whether the term human fetal membranes possess the enzymic ability to synthesize dehydroepiandrosterone (DHEA) from pregnenolone, homogenates of amnion and chorion obtained from women (n = 5, age 27-34 years) after spontaneous labor at term (37-42 weeks gestation) from uncomplicated pregnancies were incubated with [7n-3H]pregnenolone as substrate. Reverse-isotope dilution analysis gave positive identification of [3H]DHEA acetate in all incubations of viable tissues. No such metabolite was evident in control incubations with heat-denatured tissues. Virtually radiochemically pure esters under three recrystallizations were obtained with mean concentrations of between 15787 and 30137 dpm mol(-1) for amnion which was considerably higher than that of chorionic tissues at 4316-5528 dpm mol(-1). The magnitude of elevation in DHEA production by amnion was noted to be between 3.6- and 5.5-fold higher than the corresponding chorion. This study provides evidence that the fetal membranes possess 17-alpha hydroxylase and C-17, 20 lyase activities capable of synthesis of DHEA, an important androgen necessary for aromatization to estrogens in need by the developing fetus.

Expression profiles of interleukin-15 in early and late gestational human placenta and in pre-eclamptic placenta.

The presence of interleukin-15 (IL-15) mRNA in human placenta has been demonstrated previously. The present study was undertaken to investigate the expression profiles of IL-15 mRNA and protein in early and late gestational placental tissues, and also the effect of labour on its production. Levels of placental IL-15 expression were also determined in patients presenting with pre-eclampsia. An explant culture system was used to study the release of immunoreactive IL-15 by the placental tissues. Enzyme-linked immunosorbent assays were employed to quantify concentrations in the culture medium. The results showed that placental tissues from all groups released immunoreactive IL-15 into the culture medium. Moreover, the level of secretion by the term placental tissues was much higher than that by first trimester tissues. The presence of labour at term resulted in a further increase in placental IL-15 production. Reverse transcription-polymerase chain reaction (RT-PCR) was used to demonstrate the expression of IL-15 mRNA in these tissues. The results confirmed the expression of IL-15 in placenta from all the groups and the mRNA levels in the samples was highly correlated with the respective protein concentrations. Levels of both IL-15 mRNA and protein were significantly reduced in the pre-eclamptic placental tissue compared with the normal controls. The present study suggests an important role for this novel cytokine in human pregnancy.

Effect of T-helper 1 cytokines on secretion of T-helper 2 cytokines by term trophoblast cells in culture.

A successful pregnancy has been postulated to be the result of a discrete balance between T-helper 1 (Th1) and T-helper 2 (Th2) type cytokines involved in growth and development of the conceptus. The aim of the present study was to examine the effect of Th1 cytokines (interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha)) on the release of Th2 cytokines including IL-6 and IL-10 by trophoblast cells obtained from term placenta. Trophoblast cells isolated by enzymatic disaggregation and Percoll gradient fractionation were cultured in supplemented medium alone or with varying concentrations of the selected recombinant cytokines. After 48 h of incubation, samples of the culture supernatant were analyzed for the Th2 cytokines IL-6 and IL-10 using specific ELISA assays. Both IL-1 beta and TNF alpha had no effect on the cell number and viability as determined by MTT assay. IL-1 beta significantly stimulated trophoblast release of IL-6 in a dose-dependent manner (3.3-, 5.5-, 10.3- and 22.4-fold higher compared to the control at 10, 50, 100, 500 U IL-1 beta/ml respectively, p < 0.05). TNF alpha also stimulated release of IL-6 by these cells. However, the stimulation at lower concentrations was not very high and a significant (p < 0.05) stimulation was observed only at higher concentrations (1.1-, 1.3-, 2.6- and 5.9-fold higher at 500, 1000, 1500, 2000 U TNF alpha/ml respectively). In contrast, neither IL-1 beta or TNF alpha exerted any significant effect on IL-10 release by term trophoblast cells (p > 0.05). The results of this study provide evidence that production of Th2 cytokines might be under the control of different regulatory pathways.

Evidence for de novo cholesterol synthesis by term human fetal amnion and chorion: a comparative study using the reverse-isotope dilution technique.

The cholesterol biosynthetic activity was assessed using [2-(14)C]-acetate as substrate in the homogenates of amnion and chorion obtained from women (n = 6, age 26-39 years) after spontaneous labour at term (37-40 weeks of gestation) having uncomplicated pregnancies. Reverse-isotope dilution analysis gave positive identification of [(14)C]-cholesterol acetate in all incubations of viable tissues. This metabolite was not evident in heat-denatured homogenates which served as controls. The extent of enzymic conversion for amnion at 2.6 x 10(-3) to 0.19% was persistently higher than that of the chorion at 1.7 x 10(-3) to 9.0 x 10(-3)%. The results indicate that human term fetal membranes possess the full complement of enzymes to catalyze the transformation of acetate to cholesterol. This study provides evidence that fetal membranes possess the capacity for de novo cholesterol biosynthesis, the sterol being essential for steroidogenesis as well as in embryo viability during pregnancy.

Evidence for progesterone synthesis by human umbilical cord blood erythrocytes.

In order to determine whether human umbilical cord blood erythrocytes are capable of progesterone biosynthesis, sonicated preparations of plasma-free erythrocytes at the range of total cell numbers between 18.4 x 10(9) and 62.9 x 10(9) cells obtained from umbilical cord arterial and venous blood collected from normal pregnant women (n = 6, age 28-39 years) following spontaneous vaginal delivery were incubated with [7n-(3)H]-pregnenolone as substrate. The leucocyte content of the incubates was negligible (<0. 005%). Controls (n = 4, age 29-36 years; 27.6 x 10(9) to 42.7 x 10(9) erythrocytes) obtained from cord blood of normal pregnant women were heat-denatured preparations of cells. Using the reverse-isotope dilution technique, [(3)H]-progesterone was isolated and characterized yielding an overall enzymic conversion which ranged between 0.27 and 0.46%. The results indicate for the first time that cord blood erythrocytes possess the 3beta-hydroxysteroid dehydrogenase-5,4-en isomerase activity and are a source of progesterone in human pregnancy.

Increased expression of interleukin 6 in term compared to the first trimester human placental villi.

Cytokines and their specific receptors expressed at the feto-maternal interface are known to play a critical role in regulating various placental functions. Interleukin 6 (IL-6) has been shown to be produced by both decidua and the trophoblast cells of the placenta. The aim of the present study was to examine the expression profile of placental IL-6 protein and mRNA at early and late stages of gestation. Placental villi were obtained from women undergoing first trimester pregnancy termination or elective Cesarean section at term. Functionally active placental explant culture system was used to study the release of IL-6 by these tissues. IL-6 was detected in placental conditioned media of all the samples from first trimester and term group. The mean levels of IL-6 produced by term villi were found to be 5.5, 7.5 and 5-fold higher at term when compared with the first trimester at 24 h, 48 h and 72 h of culture, respectively. Expression of IL-6 mRNA was demonstrated by RT-PCR performed on total RNA isolated from these tissues. IL-6 mRNA expression was detected in both early and late gestational placental tissues. Moreover, the level of IL-6 mRNA was found to be approximately 4-fold higher at term compared with first trimester. These data are consistent with the hypothesis that levels of IL-6 production by the placenta are developmental stage-specific and suggest that expression of IL-6 in the placenta could be subjected to transcriptional regulation.

Mitochondrial gene mutations in gestational diabetes mellitus.

Mitochondrial DNA mutations have been implicated in many diseases including diabetes mellitus. Although gestational diabetes mellitus (GDM) has been suggested to have genetic determinant and to be etiologically indistinct with non-insulin-dependent diabetes mellitus (NIDDM), its association with mitochondrial gene mutations is still unknown. In this study, 137 patients with GDM and 292 non-diabetic pregnant controls were examined for mitochondrial DNA mutations from the nucleotide 3130-4260 encompassing tRNA-Leu gene and adjacent NADH dehydrogenase 1 gene by polymerase chain reaction, single-stranded conformation polymorphism, restriction fragment length polymorphism and DNA sequencing. One heteroplasmic mutation at the position of 3398 (T-C), which changed a highly conserved methionine to threonine in NADH dehydrogenase subunit 1, was identified in 2.9% GDM patients but not in the controls, indicating its association with GDM (P = 0.01). Two novel mutations, a heteroplasmic C3254A and a homoplasmic A3399T, were also found in GDM subjects, the functional meaning of which merits further investigation. G3316A and T3394C mutations implicated in NIDDM, were seen at higher frequencies in patients with GDM than the controls. Our results suggest that mitochondrial DNA mutations may contribute to the development of GDM in some patients.

Expression of C-20, 22 desmolase activity by the human fallopian tube in vitro: evidence for steroidogenesis.

With a view to establishing whether cells of the human Fallopian tubes possess the cholesterol side-chain cleavage activity, homogenates of the tubes, obtained from 6 women (39-45 years) following abdominal hysterectomy for benign conditions, were incubated with (7n-3H)-cholesterol as substrate. Controls (n=6, age 40-44 years) were homogenates heated in a boiling water bath for 10 min. Using the reverse-isotope dilution technique, (3H)-pregnenolone was isolated and characterized. No such metabolite was evident in control incubations of heat-denatured enzymes. The extent of enzymic conversion varied from 1.9 x 10(-3) to 1.3 x 10(-2)%. The results reveal for the first time the existence of an active cholesterol-specific C-20, 22 desmolase system in the viable tissues. It is suggested that there exists a potential for substantial pregnenolone synthesis in vivo. This rate-limiting steroid biosynthetic conversion provides a new dimension to the functional capacity of the Fallopian tubes in the synthesis of steroids, which may be necessary for modulating ciliary beat frequency and in maintenance of hormonal milieu essential for embryogenesis.

Association of molecular variants of luteinizing hormone with menstrual disorders.

OBJECTIVE: Luteinizing hormone (LH) promotes ovulation and luteinization of the ovarian follicle, and stimulates steroidogenesis in the ovaries. It is known to be present in different molecular forms, and secretion of abnormal LH has been implicated in menstrual disorders and infertility. The purpose of this study was to determine any association of two recently described LH variants with menstrual disorders in Singapore Chinese women. One of these variants had Trp8 to Arg8 and Ile15 to Thr15 replacements in the LH beta-subunit, while the second variant possessed Ser102 substitution for Gly102. PATIENTS: One hundred and seventy six patients with menstrual disorders and two hundred normal ovulatory women were recruited and screened for the presence of these two LH variants. METHODS: The polymerase chain reaction (PCR) products of patients were analysed by restriction fragment length polymorphism (RFLP) and the results were compared with those of normal ovulatory women and confirmed by DNA sequencing. RESULTS: Twenty one (11.9%) patients with menstrual disorders and twenty (10%) normal ovulatory women were found to carry the first variant, but its occurrence did not show any significant statistical difference between the patient and control groups (P = 0.679). However, the second variant was only detected in seven (4%) patients with menstrual disorders, and none of the normal ovulatory subjects (P = 0.005). CONCLUSIONS: the study showed that the first variant was not associated with menstrual disorders, whereas the second variant might be implicated in menstrual disorders in some Singapore Chinese women.

Expression of 3beta-hydroxysteroid dehydrogenase-5,4-en isomerase activity by infiltrating ductal human breast carcinoma in vitro.

In order to determine whether human mammary tumors could contribute to progesterone synthesis from pregnenolone in breast cancer patients, homogenates of infiltrating ductal primary breast tumors at different stages of malignancy (Stages II and III) obtained from pre- and post-menopausal patients (n = 7, age 37-66 years) were incubated with [7n-3H]pregnenolone as substrate. Controls were heated homogenates instead of fresh homogenates. With the use of reverse-isotope dilution analysis, [3H]progesterone was isolated and characterized. No such metabolite was evident in the control incubations of heat-denatured enzymes. The extent of enzymic conversion varied from 0.02 to 4.0%. The results reveal that activity of 3beta-hydroxysteroid dehydrogenase-5,4-en isomerase that metabolizes pregnenolone to progesterone can be identified with the viable homogenates. It is suggested that there exists a potential for substantial progesterone synthesis in vivo. This conversion may be of considerable clinical, therapeutic, and pathophysiological significance in the patient with breast cancer. The biological impact of this conversion should be a high priority research objective.

A delta 4-3-keto pathway for testosterone synthesis in the human spermatozoa.

The ability of human spermatozoa to metabolize pregnenolone to progesterone and progesterone to testosterone was assessed. Sonicated specimens of freshly ejaculated sperm from two groups of husbands (n = 6, age 32-38 years; n = 6, age 30-51 years) of infertile couples in the range of sperm concentration between 237.5 and 568.5, 100.1 and 248.8 millions per ejaculate, were separately incubated with [7n-3H]pregnenolone and [1,2,6,7,16,17-3H]progesterone. Using the classical reverse-isotope dilution technique the desired products [3H]progesterone and [3H]testosterone formed from the respective substrates were isolated and characterized, yielding 1.4 to 12.2% and 3.1 x 10(-2) to 2.0 x 10(-1)%. Such metabolites were not evident in the controls. The results indicate that the human spermatozoa contain the enzymes necessary for the transformation of pregnenolone to testosterone via the delta 4-3-keto route.