Human placental norepinephrine transporter mRNA: expression and correlation with fetal condition at birth

The purpose of this study was to determine the primary form of human placental norepinephrine transporter (hNET) mRNA expressed in the human placenta and to compare the level of expression in normal pregnancies and in pregnancies complicated by drug exposure or other forms of physiological derangement. We used the hNET cDNA to measure RNA extracted from placenta and examined placental RNA following complicated and uncomplicated pregnancies. To compare transporter expression and its relation to fetal condition at birth, umbilical arterial plasma catecholamine levels, umbilical arterial blood gases and placental transporter mRNA level were compared by linear regression analysis. Uncomplicated pregnancies had a higher level of placental norepinephrine transporter mRNA than complicated pregnancies. An inverse relationship between umbilical cord norepinephrine level and transporter expression was demonstrated. We conclude that placental transporter expression represents an important and newly described metabolic function of the placenta. Placental catecholamine clearance mediated via the placental NET may be important in the pathophysiology of disorders associated with placental dysfunction, impaired placental blood flow or intrauterine growth retardation. This may also explain the adverse effects of drugs, such as cocaine, which block catecholamine transport.     

Abnormal expression of type 1 plasminogen activator inhibitor and tissue factor in severe preeclampsia

Preeclampsia is a multisystemic obstetric disease of unknown etiology that is commonly associated with fibrin deposition, occlusive lesions in placental vasculature, and intrauterine fetal growth retardation. We previously reported that type 1 plasminogen activator inhibitor (PAI-1) levels are significantly increased in plasma and placenta from pregnant women with preeclampsia compared to normal pregnant women. In the present report we localize the expression of placental PAI-1 in greater detail and compare it with that of tissue factor (TF), a procoagulant molecule, and vitronectin (Vn), a PAI-1 cofactor. We also examine the expression of two cytokines, tumor necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1), in order to begin to define the underlying mechanisms responsible for the elevated levels of PAI-1 and fibrin deposits observed in placenta from preeclampsia. We demonstrate a significant increase in PAI-1, TF and TNFalpha antigen and PAI-1 and TF mRNA in placentas from preeclamptic patients. PAI-1 mRNA was increased not only in syncytiotrophoblast and infarction areas, but also in fibroblasts and in some endothelial cells of fetal vessels in placentas from preeclamptic patients. However, there was no colocalization between PAI-1, TF, Vn and TNFalpha in placental villi. The elevated TNFalpha in the placenta may induce PAI-1 and TF, and thus promote the thrombotic alterations associated with preeclampsia.     

Non-invasive mapping of placental perfusion

BACKGROUND: We aim to develop a clinical technique for the non-invasive measurement of placental perfusion, to enable early detection of intrauterine growth restriction (IUGR). Pregnancies with this complication are characterised by low placental perfusion. METHODS: We measured placental perfusion by means of perfusion-sensitive echoplanar imaging (EPI); a rapid method of making magnetic resonance images. Perfusion measurements were done on six healthy volunteers with normal pregnancies and nine with pregnancies complicated by IUGR. Perfusion maps were created to assess the relation between placental perfusion and fetal size at birth. FINDINGS: Pregnancies complicated by IUGR differed significantly from normal pregnancies in patterns of perfusion within the placenta (p<0.0001, ANOVA). Subsequent analysis showed that the proportion of placentas with low perfusion rates was higher in the IUGR group than in the normal group. A significant correlation between areas of reduced placental perfusion and fetal size was demonstrated (p=0.041, Spearman's rank correlation). INTERPRETATION: Non-invasive imaging of placental perfusion by means of EPI has potential as a clinical tool in assessing the dynamics of placental perfusion.     

Expression of cell adhesion molecules in placentae from pregnancies complicated by pre-eclampsia and intrauterine growth retardation

Platelets and neutrophils are involved in maternal placental vascular damage in pre-eclampsia. Recruitment of these cells is probably mediated by cell adhesion molecules expressed at the uteroplacental bed. It remains controversial as to whether platelets and neutrophils mediate damage to trophoblast or villous vasculature. The purpose of this study was to determine the expression of cell adhesion molecules in placentae from normal pregnancies and pregnancies complicated by pre-eclampsia and intrauterine growth retardation (IUGR). Immunostaining for platelet endothelial cell adhesion molecule (PECAM) and intercellular adhesion molecule-1 (ICAM-1) was localized mainly to the endothelium of stem villi, intermediate villi, terminal villi and decidual vessels. Scattered staining for ICAM-1 was also evident in the stroma and fetal membranes. The endothelium of stem villi, intermediate villi and terminal villi were all negative for vascular cell adhesion molecule-1 (VCAM-1) and E-Selectin. PECAM, ICAM-1 and ICAM-2 mRNA were all detectable in normal placentae using northern blotting analysis whereas mRNA for E-Selectin and VCAM-1 were both undetectable. There were no differences in cell adhesion molecule immunostaining or mRNA expression in placentae from pregnancies complicated by pre-eclampsia and IUGR inconclusion, expression of cell adhesion molecules in placentae from pre-eclampsia and IUGR are consistent with a normal physiological role in vascular function.     

Circulating thyroid hormone concentrations and placental thyroid hormone receptor expression in normal human pregnancy and pregnancy complicated by intrauterine growth restriction (IUGR)

Thyroid hormones are critical to growth and development of the human fetus. Abnormal placental development, a major cause of intrauterine growth restriction (IUGR), is associated with a high perinatal mortality and morbidity. Thyroid status has been postulated to play a role in the pathogenesis of such morbidity. In the present study, we have investigated fetal thyroid function and placental expression of thyroid hormone receptor (TR) alpha and beta variants during normal human pregnancy and in pregnancy associated with IUGR. Measurement of free thyroid hormones and TSH concentrations revealed significant rises in free T4 and free T3 between the second and third trimesters of normal pregnancy. Serum concentrations of free T4 and free T3 were lower in fetuses affected by IUGR, although serum TSH levels were not significantly different. Immunocytochemistry demonstrated the presence of TR alpha1, alpha2, and beta1 proteins within the nuclei of trophoblast and stromal placental cells. Immunostaining for these TR variants increased with increasing gestation in normal placenta. Comparison of IUGR placental samples with normal samples revealed greater immunostaining for TR alpha1, alpha2, and beta1 variants in IUGR. Examination of pretranslational expression of TR alpha1, alpha2, beta1, and beta2 variants by semiquantitative RT-PCR revealed increasing expression of TR alpha1, alpha2, and beta2 messenger RNAs with increasing gestation in normal pregnancy, which "mirrored" post-translational expression. However, and in contrast, there were no significant differences in expression of TR messenger RNAs in normal and IUGR placenta. The present findings of reduction in serum free thyroid hormones and increased expression of TR alpha and beta proteins in association with IUGR highlight the potential importance of thyroid status in influencing long-term fetal outcome in this condition.     

Nitric oxide synthase in human placenta and umbilical cord from normal, intrauterine growth-retarded and pre-eclamptic pregnancies

1. It has been suggested that a deficiency of nitric oxide (NO) may explain many of the pathophysiological features of pre-eclampsia (PE) and intra-uterine (foetal) growth retardation (IUGR). To elucidate further the role of NO in the pathophysiology of pregnancy we have determined the relative amount and activity of NO synthase (NOS) in first trimester and normal-term placental tissues, as well as in the placenta and umbilical cord in pregnancies complicated by PE and IUGR, using NG-nitro-L-[2,3,4,5(-3)H]-arginine ([3H]-L-NOARG) binding, quantitative in vitro autoradiography, [3H]-arginine to [3H]-citrulline conversion and Western blotting. 2. Specific, high affinity (KD = 38 nM) [3H]-L-NOARG binding was demonstrated in the villous trophoblast of normal-term placentae. Binding was calcium-independent, stereoselective and exhibited a rank order of inhibition by NOS inhibitors and substrate (L-NOARG > or = L-NMMA > or = 7-NI > L-NAME > L-Arg > or = L-NIO > ADMA). 3. [3H]-L-NOARG binding density and NOS activity were both significantly greater in placental tissues from first trimester and PE or IUGR complicated pregnancies compared to normal-term placentae. 4. Western blotting, using an endothelial NOS peptide antiserum, demonstrated a approximately 140 KDa protein band in placental extracts and indicated that the amount of immunoreactive material was significantly greater in first trimester compared to normal-term placentae. 5. Specific [3H]-L-NOARG binding was also localized to the endothelial lining of umbilical arteries and veins, binding density being greater in the artery than the vein. [3H]-L-NOARG binding to the umbilical artery endothelium was significantly lower in PE and IUGR complicated pregnancies compared to normal-term controls. 6. The role of trophoblast-derived NO in human placental pathophysiology remains to be established, but differences in the amount of placental [3H]-L-NOARG binding, NOS activity and immunoreactive material indicate that expression of NOS in the villous trophoblast falls during pregnancy. Conversely, the apparent reduction in NOS in the umbilical artery endothelium in PE and IUGR complicated pregnancies may be indicative of endothelial dysfunction.     

Lipid and lipoprotein concentrations in pregnancies complicated by intrauterine growth restriction

Previous studies have shown that in preeclampsia, plasma lipids climb substantially above levels seen in normal pregnancies. Such lipid changes may play a role in the endothelial damage characteristic of preeclampsia. Pregnancies complicated by intrauterine growth restriction (IUGR), without preeclampsia, have similar placental pathology to preeclampsia despite the absence of the maternal systemic manifestations of hypertension and proteinuria. The aim of this study was to perform a cross-sectional study of lipid and lipoprotein concentrations in the third trimester, from normal pregnancies, and those complicated by IUGR without preeclampsia. Our hypothesis was that, in contrast to the exaggerated lipid changes seen in preeclampsia, lipid and lipoprotein concentrations in IUGR would be similar to those of matched healthy pregnant controls. Fasting blood samples for lipids and lipoprotein fractions were taken in the third trimester, from eight women with IUGR; and eight women with uncomplicated pregnancies, matched as a group for age, booking weight, parity, and gestational age at sampling. There were no significant differences (P > 0.05) in the median concentrations of triglyceride, high-density lipoprotein, and very-low-density lipoprotein 1 (VLDL1), between cases and controls. However, women with IUGR pregnancies had significantly lower cholesterol [4.95 mmol/L (3.35-7.10) vs. 7.47 (5.75-8.45); median (range) for IUGR patients and controls, respectively; P < 0.01], low-density lipoprotein (LDL)-cholesterol [2.45 mmol/L (0.95-3.60) vs. 4.25 (3.35-5.60); P < 0.01], VLDL2 mass [59.0 mg/dL (37-87) vs. 103.0 (64-168); P < 0.01], intermediate-density lipoprotein mass [56.0 mg/dL (31-110) vs. 125.6 (91-157); P < 0.01], and total LDL mass [221.0 mg/dL (104-237) vs. 380.3 (267-534); P < 0.01]. In addition, it was noteworthy that, with respect to LDL-cholesterol and total LDL mass, there was little or no overlap in the ranges of concentrations measured between cases and controls. Because VLDL2 and intermediate-density lipoprotein are the synthetic precursors to LDL in the circulation, their significantly lower median concentrations imply a failure of appropriate LDL synthesis in IUGR pregnancies. Whatever the mechanism, if our results are confirmed in larger studies and longitudinal investigations, then LDL-cholesterol measurements (when LDL-cholesterol fails to rise appropriately or is low in the third trimester) may be of use in identifying mothers with, or at risk of, a pregnancy complicated by IUGR.     

Differential distribution of endothelin receptor subtypes in placentae from normal and growth-restricted pregnancies

The endothelins (ETs) are potent vasoconstrictor peptides that bind to two distinct receptors, ETA and ETB. This study compares the localization of ETA and ETB receptors in placentae complicated by intrauterine growth retardation (IUGR) and abnormal umbilical Doppler waveform, gestationally matched controls, fetuses that were small for gestational age (SGA), and normal term placentae. Quantitative autoradiography was performed using ETA and ETB subtype-selective ligands. Both ETA and ETB receptors were expressed in the human placenta. Gestational and fetal size effects on the receptor density within stem villi were found, but no effect of abnormal placental blood flow could be demonstrated. A distinct spatial distribution of receptor subtypes within the placenta was observed. Smooth muscle cells expressed both receptors with ETA expression predominant in the proximal regions of the villous tree and ETB abundant in the periphery and decidua. Both receptors were also expressed at lower density on paravascular stromal cells in stem villi. Although these data do not demonstrate aberrant localization of ET receptors in IUGR and SGA placentae, the spatially distinct distribution of ET receptors in the human placenta suggests that ETs play a role in modulation of placental blood flow.     

Biochemical markers of trisomy 21 and the pathophysiology of Down's syndrome pregnancies

Using biochemical and immunocytochemical methods, we have investigated endogenous levels of various markers in tissues obtained from 67 Down's syndrome pregnancies after therapeutic abortion in the second trimester and in corresponding tissues from unaffected abortuses. Alpha-fetoprotein (AFP), intact and free beta human chorionic gonadotrophin (hCG), pregnancy-specific beta-1 glycoprotein (SP-1), placental alkaline phosphatase (PALP), pregnancy-associated plasma protein A (PAPP-A), and gamma glutamyl transferase (GGT) were investigated in placental tissue; AFP and GGT in fetal liver; and GGT in fetal intestine. The results indicate that maternal serum levels of placental products reflect those found in the placenta: intact hCG, free beta hCG, and SP-1 levels were elevated in Down's syndrome pregnancies, while PAPP-A and PALP levels were little changed. This suggests that membrane passage of these markers is not affected but there is altered synthesis of hCG and SP-1. AFP levels were strikingly elevated in placental homogenates and unchanged in liver homogenates from Down's syndrome pregnancies, while the levels in maternal serum were reduced, pointing to a possible transport defect specific to AFP. GGT levels were high in placenta and liver from Down's syndrome pregnancies but low in fetal intestine.     

Proteases and their inhibitors are indicative in gestational disease

OBJECTIVE: To assess whether various proteolytic factors which are involved in trophoblast invasion show different concentrations in plasma and placenta of patients with HELLP syndrome, pre-/eclampsia and highly pathological Doppler flow measurements but without additional complications (hpD). DESIGN: Case control and observational study; 18 women with HELLP syndrome, 21 with pre-/eclampsia, 13 with hpD, as well as healthy pregnant women (matched pairs); statistical analysis: sign test and Wilcoxon test. RESULTS: Urokinase-type plasminogen activator (uPA), uPA receptor, tissue-type plasminogen activator (tPA), plasminogen activator inhibitor 1 (PAI-1), matrix metalloproteinases MMP-8, MMP-9 and tissue inhibitor of metalloproteinases TIMP-1 were measured by ELISA. PAI-1 plasma levels are significantly elevated in all three groups studied. In HELLP syndrome, tPA and TIMP-1 are also elevated, and in patients with hpD, MMP-8 is increased, whereas MMP-9, and TIMP-1 are lower. In placenta extract, only pre-/eclampsia shows reduced MMP-9 concentrations. CONCLUSIONS: The increased frequency of small-for-gestational-age infants observed in all three study groups is an expression of impaired placental implantation and remodelling processes. These disturbances manifest themselves in the form of changes in some of the factors in plasma and placenta extract that are involved in these processes.     

Angiotensin II regulates the expression of plasminogen activator inhibitor-1 in cultured endothelial cells. A potential link between the renin-angiotensin system and thrombosis

Plasminogen activator-inhibitor C-1 (PAI-1) plays a critical role in the regulation of fibrinolysis, serving as the primary inhibitor of tissue-type plasminogen activator. Elevated levels of PAI-1 are a risk factor for recurrent myocardial infarction, and locally increased PAI-1 expression has been described in atherosclerotic human arteries. Recent studies have shown that the administration of angiotensin converting enzyme inhibitors reduces the risk of recurrent myocardial infarction in selected patients. Since angiotensin II (Ang II) has been reported to induce PAI-1 production in cultured astrocytes, we have hypothesized that one mechanism that may contribute to the beneficial effect of angiotensin converting enzyme inhibitors is an effect on fibrinolytic balance. In the present study, we examined the interaction of Ang II with cultured bovine aortic endothelial cells (BAECs) and the effects of this peptide on the production of PAI-1. 125I-Ang II was found to bind to BAECs in a saturable and specific manner, with an apparent Kd of 1.4 nM and Bmax of 74 fmol per mg of protein. Exposure of BAECs to Ang II induced dose-dependent increases in PAI-1 antigen in the media and in PAI-1 mRNA levels. Induction of PAI-1 mRNA expression by Ang II was not inhibited by pretreating BAECs with either Dup 753 or [Sar1, Ile8]-Ang II, agents that are known to compete effectively for binding to the two major angiotensin receptor subtypes. These data indicate that Ang II regulates the expression of PAI-1 in cultured endothelial cells and that this response is mediated via a pharmacologically distinct form of the angiotensin receptor.     

Telomerase activity in human chorionic villi and placenta determined by TRAP and in situ TRAP assay

Telomerase activity (TA) was analysed in human chorionic villi and placenta in normal and abnormal pregnancy using the telomeric repeat amplification protocol (TRAP) and in situ TRAP assay. Twenty chorionic villi specimens and 25 placenta specimens from normal pregnancies were examined as well as placenta specimens from 10 cases of intrauterine growth retardation (IUGR; nine asymmetric and one symmetric). TA was detected in 18 of the 20 (90 per cent) chorionic villi specimens and in 18 of the 25 (72 per cent) placenta specimens from normal pregnancy. However, no or only weak TA was exhibited in the placenta specimens of the nine asymmetric IUGR cases. In situ TRAP assay detected TA in trophoblastic cells from normal pregnancy, but not in trophoblastic cells from cases of asymmetric IUGR.     

Cocaine-sensitive sigma-receptor and its interaction with steroid hormones in the human placental syncytiotrophoblast and in choriocarcinoma cells

The expression and ligand binding characteristics of sigma-receptors in human placental syncytiotrophoblast and choriocarcinoma cells were investigated using haloperidol as a ligand. Haloperidol bound to purified placental brush border membranes with high affinity; the apparent dissociation constant for the process was about 3 nM. These binding sites were not related to dopamine (D2) and serotonin (5-HT2) receptors nor to serotonin and norepinephrine transporters. The ligands of sigma-receptors [3.g. (+)-3-(3-hydroxyphenyl)N-(1-propyl)piperidine, 1,3-di-(2-tolyl)guanidine, clorgyline, rimcazole, and dexromethorphan] were very potent in competing with haloperidol for the binding sites. The binding sites were detected not only in the brush border membrane, but also in intracellular membranes. The rank order of potency of various sigma-receptor ligands to inhibit haloperidol binding indicated that placental sigma-receptors belong to the sigma 1 subtype. Cocaine and its analog RTI-55 [2 beta-carbomethoxy-3 beta-(4-iodophenyl-) tropane] inhibited the binding of haloperidol to placental membranes with appreciable potency. The steroid hormones, progesterone and testosterone, were also potent inhibitors, and the inhibition constant for progesterone was 0.3 microM, a concentration much smaller than that found in plasma during pregnancy. The inhibition was competitive. beta-Estradiol and a number of other steroids were relatively much weaker inhibitors than progesterone and testosterone. Phenytoin and neuropeptide-Y did not interact with sigma-receptors in placenta. The choriocarcinoma cell line JAR was also found to express sigma-receptors in the plasma membrane as well as in intracellular membranes. The characteristics of the receptors in this cell were qualitatively similar to those of the receptors in normal placenta, including subtype identity and interaction with cocaine and progesterone. Interestingly, however, all sigma-receptor ligands interacted with the receptors in the JAR cell with much higher affinity than with the receptors in normal placenta. It is concluded that the placental syncytiotrophoblast and choriocarcinoma cells express cocaine-sensitive sigma-receptors and that progesterone is most likely an endogenous ligand for these receptors.     

Prevention of spinal anesthesia-induced hypotension in the elderly: comparison between preanesthetic administration of crystalloids, colloids, and no prehydration

The expression of tissue transglutaminase (tTG) was studied during the formation of the normal human placenta and in molar pregnancies and choriocarcinoma, in order to correlate its expression with the functional characteristics of the recognized trophoblast cell types. tTG expression was found to be developmentally regulated. Before 6-7 weeks' gestation, only the chorionic villous cytotrophoblast expresses tTG. Thereafter the overlying syncytiotrophoblast becomes positive. tTG expression is gradually downregulated in the intermediate trophoblast cells emerging from the tips of the chorionic villi invading the uterine tissue. In the decidual wall, the intermediate trophoblast does not express tTG, whereas scattered syncytial cells, the placental bed giant cells, express tTG. Villi from complete hydatidiform mole (CHM) show tTG expression in both the cyto- and the syncytiotrophoblast. The intermediate trophoblast cells from CHM show heterogeneous tTG expression, with a majority of negative cells, whereas extravillous syncytia always express tTG. In choriocarcinoma, the tumour cells show heterogeneous tTG expression, with a majority of positive cells. Analysis of tTG protein and mRNA in placental extracts by Western and Northern blotting did not provide evidence for expression of the truncated form of tTG found in some cell types. The regulated expression of tTG in the normal placenta suggests that the enzyme is involved in important trophoblastic functions and may participate in the control of invasion.     

Effects of sodium nitroprusside on hemodialysis-induced platelet activation

Plasminogen activator inhibitor-2 (PAI-2), a member of the serpin gene family, is thought to serve as a primary regulator of plasminogen activation in the extravascular compartment. High levels of PAI-2 are found in keratinocytes, monocytes, and the human trophoblast, the latter suggesting a role in placental maintenance or embryo development. The primarily intracellular distribution of PAI-2 also may indicate a unique regulatory role in a protease-dependent cellular process such as apoptosis. To examine the potential functions of PAI-2 in vivo, we generated PAI-2-deficient mice by gene targeting in embryonic stem cells. Homozygous PAI-2-deficient mice exhibited normal development, survival, and fertility and were also indistinguishable from normal controls in response to a bacterial infectious challenge or endotoxin infusion. No differences in monocyte recruitment into the peritoneum were observed after thioglycollate injection. Epidermal wound healing was equivalent among PAI-2 -/- null and control mice. Finally, crossing PAI-2 -/- with PAI-1 -/- mice to generate animals deficient in both plasminogen activator inhibitors failed to uncover an overlap in function between these two related proteins.