Hypoxia blocks 11beta-hydroxysteroid dehydrogenase type 2 induction in human trophoblast cells during differentiation by a time-dependent mechanism that involves both translation and transcription.

The present study was undertaken to determine (1) if hypoxia-induced down-regulation of placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2; encoded by HSD11B2 gene) activity and protein in human trophoblast cells during in vitro differentiation was mediated at the level of HSD11B2 gene transcription; and (2) whether the reduced placental 11beta-HSD2 in pregnancies complicated with fetal growth restriction (FGR) was a consequence of intrinsic abnormalities in trophoblast cells. Trophoblast cells were isolated from uncomplicated pregnancies and those complicated with FGR at term, and cultured for up to 72 h under normoxic (20% oxygen) or hypoxic (1% oxygen) conditions. Under normoxia, 11beta-HSD2 activity and protein increased progressively over the 72 h culture period, which was accompanied by a corresponding rise in 11beta-HSD2 mRNA. As demonstrated previously, hypoxia blocked the increase in levels of both 11beta-HSD2 activity and protein within the first 24h. In contrast, although hypoxia also prevented the rise in 11beta-HSD2 mRNA, it did not do so until 48 h. This time-dependent effect of hypoxia on placental 11beta-HSD2 activity/protein and mRNA suggests a dual mechanism of action whereby hypoxia may induce a rapid down-regulation of 11beta-HSD2 protein synthesis, which occurs initially at the level of translation, and later extends to the level of transcription. Indeed, transient transfection studies demonstrated that hypoxia diminished HSD11B2 promoter activity. When trophoblast cells isolated from FGR placentas were cultured and allowed to differentiate under the same conditions, they not only exhibited a similar pattern of 11beta-HSD2 activity and mRNA expression but also responded to hypoxia similarly to those from normal placentas. This suggests that the reduced placental 11beta-HSD2 in FGR is not due to intrinsic abnormalities in trophoblast cells, but likely a result of extrinsic factors associated with FGR.

The expression of 11 beta-hydroxysteroid dehydrogenase type 2 is induced during trophoblast differentiation: effects of hypoxia.

The intracellular enzyme 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) catalyzes the unidirectional conversion of bioactive glucocorticoids to their inert metabolites. In the human placenta, 11 beta-HSD2 is highly expressed in syncytiotrophoblasts, although cytotrophoblasts also express this enzyme at lower levels. Given that cytotrophoblasts will differentiate into syncytiotrophoblasts in vivo and in vitro, the present study was designed to examine the hypothesis that the expression of 11 beta-HSD2 is induced during in vitro trophoblast differentiation. When Percoll-purified human cytotrophoblast cells were cultured under standard (20% oxygen) conditions, they aggregated and fused to form syncytiotrophoblasts. Within the first 24 h during differentiation, levels of 11 beta-HSD2 protein and activity were increased by 2- to 3-fold, but they did not increase further thereafter. However, when the cells were exposed to hypoxic (1% oxygen) conditions, both the induction of 11 beta-HSD2 and trophoblast differentiation were prevented. Taken together, these results demonstrate for the first time that the expression of 11 beta-HSD2 is induced early during trophoblast differentiation, and hypoxia prevents this induction, indicating that placental 11 beta-HSD2 expression is subjected to regulation by the local oxygen environment. If placental villi respond to hypoxia in a similar fashion in vivo, the present findings would suggest that hypoxia might be a factor contributing to the previously reported decreases in placental 11 beta-HSD2 in pregnancies complicated by intrauterine growth restriction and preeclampsia.

ATP stimulates human placental 11beta-hydroxysteroid dehydrogenase type 2 activity by a novel mechanism independent of phosphorylation.

The human placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) is believed to play a key role in fetal development since this enzyme protects the fetus from exposure to high levels of maternal cortisol by virtue of converting maternal cortisol to its inert metabolite cortisone. The present study was undertaken to examine the effect of ATP on 11beta-HSD2 activity in human placental microsomes. Enzyme activity, reflected by the rate of conversion of cortisol to cortisone, was stimulated more than six-fold by 0.5 mM ATP (EC(50) = 0.2 mM). Such stimulation appears to be mediated through a novel mechanism independent of ATP-induced phosphorylation of the reaction components since AMP-PNP, a non-hydrolyzable analogue of ATP, was equally effective. The ATP-induced stimulation of 11beta-HSD2 activity is adenine nucleotide specific in that a similar stimulation was observed with ADP and AMP but not with CTP, GTP, or UTP. Furthermore, ATP increased the maximal velocity (V(max)) of the 11beta-HSD2 catalyzed conversion of cortisol to cortisone without altering the apparent K(m) of 11beta-HSD2 for cortisol, suggesting that ATP may stimulate enzyme activity by interacting with the enzyme at a site other than that involved in substrate binding. In conclusion, the present study has identified ATP as a novel regulator of human placental 11beta-HSD2 in vitro. It is conceivable that intracellular ATP may have a profound effect on 11beta-HSD2 function in vivo.

Calcium inhibits human placental 11beta-hydroxysteroid dehydrogenase type 2 activity.

The effect of Ca2+ on the conversion of cortisol to its inert metabolite cortisone, the reaction catalyzed by the microsomal enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), was investigated in human placental microsomes. Placental microsomal 11beta-HSD2 activity, as determined by the rate of conversion of cortisol to cortisone, was inhibited up to 50% by increasing free Ca2+ concentrations from 22 to 268 nM. The Ca2+-induced inhibition was reversible since chelation of endogenous Ca2+ with EGTA increased 11beta-HSD2 activity up to 200%. Ca2+ decreased the maximal velocity (Vmax) of the 11beta-HSD2 catalyzed conversion of cortisol to cortisone without altering the Km of 11beta-HSD2 for cortisol, indicating that Ca2+ modulates the catalytic efficiency rather than the substrate binding of 11beta-HSD2. Moreover, the Ca2+-induced inhibition does not appear to involve altered cofactor (NAD+) binding since the inhibition of microsomal 11beta-HSD2 activity by a sub-maximal concentration of free Ca2+ was not overcome by increasing the concentration of NAD+. These findings in the microsomes were then extended to an intact cell system, JEG-3 cells, an established model for human placental trophoblasts. In these cells, an increase in cytosolic free Ca2+ concentration ([Ca2+]i) elicited by a known physiological stimulus, PGF(2alpha), was accompanied by a 40% decrease in the level of 11beta-HSD2 activity. Furthermore, the PGF(2alpha)-induced inhibition of 11beta-HSD2 activity was abrogated when increases in [Ca2+]i were blocked with the intracellular Ca2+ chelator, BAPTA. Collectively, these results demonstrate for the first time that Ca2+ inhibits human placental 11beta-HSD2 activity by a post-translational mechanism not involving substrate or cofactor binding.

Prostaglandins and leukotriene B4 are potent inhibitors of 11beta-hydroxysteroid dehydrogenase type 2 activity in human choriocarcinoma JEG-3 cells.

The 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) is responsible for the inactivation of glucocorticoids. This is the predominant isozyme in the human placenta, where it is proposed to protect the fetus from high levels of maternal cortisol. In the present study, we examined the effects of eicosanoids on the activity of 11beta-HSD2 in human choriocarcinoma JEG-3 cells, a well-established model for placental trophoblasts. Treatment of JEG-3 cells for 24 h with either prostaglandin (PG) E2 or F2alpha attenuated 11beta-HSD2 activity ( approximately 40%). Paradoxically, indomethacin, an inhibitor of cyclooxygenases, inhibited (approximately 40%) rather than stimulated the activity of this enzyme. This indicated that the arachidonic acid metabolism may be diverted to other pathway(s), the products of which may inhibit 11beta-HSD2 activity. To determine whether the lipoxygenase pathways were involved, the cells were treated with nordihydroguaretic acid (NDGA), a blocker of all three (5-, 12-, and 15-) lipoxygenases. NDGA caused a 3-fold increase in 11beta-HSD2 activity. To further delineate which specific lipoxygenase pathway was involved, the cells were incubated with zileuton, a selective inhibitor of 5-lipoxygenase. This resulted in a similar increase in 11beta-HSD2 activity, suggesting that the products of this pathway (e.g., leukotrienes) may be involved. Given that leukotriene B4 (LTB4) is the most biologically active product of the 5-lipoxygenase pathway, we treated the cells with LTB4, which inhibited 11beta-HSD2 activity in a time- and dose-dependent manner with a maximal effect (60% reduction) at 10 nM for 9 h. Semiquantitative reverse transcription-polymerase chain reaction analysis revealed that 11beta-HSD2 mRNA levels were not altered by the addition of LTB4, PGE2, or PGF2alpha, indicating an effect at the posttranscriptional level. In conclusion, these results demonstrate that prostaglandins and LTB4 are potent inhibitors of 11beta-HSD2 activity in JEG-3 cells, suggesting that placental 11beta-HSD2 activity is modulated by these locally produced eicosanoids. This is the first time that the products of arachidonic acid metabolism have been found to regulate the activity of 11beta-HSD2.

Retinoic acid stimulates the expression of 11beta-hydroxysteroid dehydrogenase type 2 in human choriocarcinoma JEG-3 cells.

The syncytiotrophoblasts of the human placenta express high levels of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), the enzyme responsible for the inactivation of glucocorticoids. It has been proposed that the placental 11beta-HSD2 serves as a barrier to protect the fetus from high levels of maternal cortisol. To examine the hypothesis that nutritional signals regulate the expression of 11beta-HSD2 in placental syncytiotrophoblasts, we investigated the effects of retinoic acids (RAs), the major metabolites of vitamin A, on the expression of 11beta-HSD2 using human choriocarcinoma JEG-3 cells as a model. This trophoblast-like cell line displays a number of functional similarities to the syncytiotrophoblast. Treatment for 24 h with all-trans RA (1-1000 nM) resulted in a dose-dependent increase in 11beta-HSD2 activity with a maximal effect (increase to 3-fold) at 100 nM. The effect of all-trans RA (100 nM) was also time-dependent in that the effect was detectable at 6 h and reached its maximum by 48 h. Similar increases in 11beta-HSD2 activity were observed when the cells were treated with 9-cis RA. Results from semi-quantitative reverse transcription-polymerase chain reaction demonstrated that there was a corresponding increase in 11beta-HSD2 mRNA after RA treatment. Moreover, treatment with actinomycin D (100 ng/ml) abrogated the increase in 11beta-HSD2 mRNA induced by RA, indicating an effect on transcription. In conclusion, the present study has demonstrated for the first time that RA, at physiological concentrations, induces 11beta-HSD2 gene expression and enzyme activity in JEG-3 cells. If this occurs in vivo, the present finding suggests that high expression of 11beta-HSD2 in the human placenta may be maintained, at least in part, by dietary intake of vitamin A.

Reproducibility of brachial ultrasonography and flow-mediated dilatation (FMD) for assessing endothelial function.

BACKGROUND: High-resolution brachial artery ultrasonography is used to study vasodilator response induced by physiologic reactive hyperaemia. We examined the reproducibility of measuring flow-mediated dilatation (FMD) on two occasions. AIMS: To determine the degree of variability of this technique in our vascular laboratory for the design of clinical research studies. METHODS: Nineteen subjects were studied on two separate occasions using an Acuson 128 ultrasound device and a 7.0 MHz linear array transducer. Reactive hyperaemia was induced in the brachial artery by inflation and release of a blood pressure cuff. Nitrate-induced dilatation was assessed in 11 of the 19 subjects. Measurements were made by two observers blinded to subject details. RESULTS: The 11 subjects given sublingual GTN during the first ultrasound study had a mean nitrate-induced dilatation of 20.7% (sd 9.6). The mean vessel diameter of 3.78 mm (sd 0.7) at rest and 3.89 mm (sd 0.7) during reactive hyperaemia yielded a mean FMD of only 3.0% (sd 2.7). The mean difference in FMD within-observers was 0.13% (sd 2.07), between-observers 0.06% (sd 2.17) and between-studies was 0.57% (sd 6.83). CONCLUSIONS: The reproducibility of FMD measured by brachial artery ultrasound was poor and likely to render the measurements inaccurate for clinical research in our hands. Between-study variation contributed the largest proportion of total study variability. We suggest that investigators using this technique conduct their own careful reproducibility studies in order to avoid the misinterpretation of 'negative' studies.

Cardiac event recorders yield more diagnoses and are more cost-effective than 48-hour Holter monitoring in patients with palpitations. A controlled clinical trial.

OBJECTIVE: To compare the diagnostic yield and cost-effectiveness of transtelephonic event monitors with those of Holter monitoring in patients with intermittent palpitations. DESIGN: Randomized crossover trial. SETTING: Diagnostic service of a teaching hospital and surrounding primary care practices. PATIENTS: 43 patients with previously uninvestigated palpitations who were referred for Holter monitoring. MEASUREMENTS: Patients were randomly allocated to receive an event monitor or 48-hour Holter monitor and then to receive the other device. Event monitors were used for 3 months or until two recordings were obtained while symptoms occurred. The main end point was an electrogram recorded during symptoms. The incremental cost-effectiveness of obtaining a diagnostic rhythm strip from event monitors was compared with that of Holter monitoring. RESULTS: The mean (+/- SD) patient age was 45 +/- 19 years; 37 patients (88%) were women. Event monitors were twice as likely to provide a diagnostic rhythm strip electrocardiogram during symptoms as 48-hour Holter monitoring (29 patients [67%] and 15 patients [35%], respectively; P < 0.001). Event monitors detected 8 patients (19%) with clinically important arrhythmias (6 patients with supraventricular tachycardia and 2 with atrial fibrillation or flutter), whereas the Holter monitors detected no significant arrhythmia (P < 0.005). With the event monitors, most patients transmitted an electrocardiogram recording by 6 weeks. Event monitors were dominant and therefore more cost-effective than 48-hour Holter monitoring, resulting in a cost savings of $213 for each additional diagnostic rhythm strip obtained during symptoms. CONCLUSIONS: Holter monitoring is a poor diagnostic test for intermittent palpitations. Event recorders provide better data and are more cost-effective.

The Psychological Maltreatment Rating Scales.

Psychological maltreatment is gaining recognition as one of the core concepts in child welfare, however, its utility has been limited by definitional problems and the absence of operationalized and validated instruments. These Psychological Maltreatment Rating Scales (PMRS) were developed for assessing psychological maltreatment in mother-child interaction, and were used to rate the videotaped interaction of 49 high-risk mother-child dyads and make predictions of child protective service involvement with the dyads. These predictions are compared with predictions based upon mothers' personal resources and social support. Results show that the PMRS is a moderately reliable and valid measure of psychologically maltreating and prosocial parental behavior that can discriminate between maltreating and comparison parents, and is a more effective predictor than maternal measures. Three factors of parenting emerged from an exploratory factor analysis: emotional abuse, and two factors of positive parenting. Psychological abuse was the presence of hostile behavior, and psychological neglect the absence of positive parenting.