Maternal tissue blood flow and oxygen saturation in pre-eclampsia and intrauterine growth restriction.

OBJECTIVE: To investigate the hypothesis that impaired maternal tissue perfusion occurs in pre-eclampsia and intrauterine growth restriction (IUGR) and this correlates with maternal tissue oxygenation. STUDY DESIGN: Strain gauge plethysmography was used to compare maternal calf blood flow during the third trimester in 16 women with pre-eclampsia, 6 women with IUGR and 16 normal pregnant controls. A Mediaid iPOX pulse oximeter was used to measure maternal tissue oxygenation in the three groups and these were compared with tissue blood flow. RESULTS: Maternal tissue blood flow was significantly reduced in pre-eclampsia compared to the two other groups (p=0.003). Blood flow was significantly reduced in pre-eclampsia compared to IUGR (p=0.03). However there was no difference in blood flow between normal pregnancy and IUGR groups (p=0.76). No significant difference was noted in maternal tissue oxygenation between the normal pregnancy, pre-eclampsia and IUGR groups (mean±S.E.M. [97.13±0.4, 96.69±0.33, 97.83±0.47 respectively], p=0.26). No correlation was noted between blood flow and tissue oxygenation in the three groups of women. CONCLUSION: We have demonstrated that reduced maternal resting tissue blood flow present in women with pre-eclampsia is not seen in women with IUGR and the reduction in blood flow in pre-eclampsia is not associated with changes in maternal tissue oxygenation.

Placental angiogenin inhibitor (ribonuclease inhibitor), a novel gene in pre-eclampsia.

There is convincing evidence that imbalance between angiogenic and anti-angiogenic factors play an important role in the pathophysiology of pre-eclampsia. Angiogenin, a member of the RNase A super family, is a potent inducer of angiogenesis and serum levels are shown to be elevated in pre-eclampsia. We hypothesize that placental expression of angiogenin inhibitor which binds and blocks the activity of angiogenin is altered in pre-eclampsia and may play a role in its pathophysiology. Placental expression of angiogenin inhibitor was measured in term placentae of 15 women with preeclampsia and 16 normal pregnant controls. The women were matched for age, parity and gestational age. Placental tissue was collected immediately after delivery and stored at -80°C until studied. Angiogenin inhibitor gene expression was measured using real-time quantitative polymerase chain reaction (rt-QPCR). The results were standardized using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference gene. The mRNA expression of angiogenin inhibitor gene was significantly increased in preeclamptic placentae compared to normal pregnant controls [0.44 (0.174-1.048) versus 0.091 (0.029-0.301), median and interquatile range, p=0.027 for pre-eclampsia and normal controls respectively]. There was no correlation between angiogenin inhibitor gene expression and maternal age, blood pressure, platelet count, gestation age, birth weight of the baby in pre-eclampsia and normal pregnancy. This study showed that placental expression of the angiogenin inhibitor gene is significantly increased in pre-eclampsia and may play a role in its pathophysiology.

A novel hysteroscopic technique for the accurate biopsy of decidua parietalis and basalis.

BACKGROUND: Discrepancies in the results from studies of early events in human trophoblast invasion of decidua have been acknowledged and are attributed largely to deficiencies in the accuracy of sampling of the decidual tissue used in the research. We describe a novel technique of biopsy of decidua parietalis and basalis that overcomes the issue of accuracy of site of the biopsy. METHODS & RESULTS: The technique is applicable to pregnancies undergoing first trimester surgical termination. Following cervical dilatation, a rigid hysteroscope is introduced into the cervical canal. The pressure of the saline distending medium shears the membranes of the gestation sac away from the decidua parietalis, leaving the pregnancy suspended at the site of the early placenta (the decidual basalis). Under direct vision a biopsy forceps is used to sample the decidua parietalis, and then the forceps is introduced beneath the gestation sac to sample the decidua basalis. Morphological and immunohistochemical studies have confirmed the accuracyof site and adequacy of the samples, with a 40% myometrial spiral artery success rate. CONCLUSION: This is a simple novel technique of decidual biopsy under direct vision which allows for high accuracy of the site of biopsy material, and therefore has the potential to revolutionize research on trophoblast-decidua interactions during the critical early stages of human pregnancy.

Gene regulation of neurokinin B and its receptor NK3 in late pregnancy and pre-eclampsia.

Elevated circulating levels of the tachykinin, neurokinin B (NKB), have been observed in women with pre-eclampsia during the third trimester of pregnancy. Currently, the molecular mechanisms responsible for these increased levels remain unknown. To understand the molecular regulation, we have compared the differences in gene expression of the tachykinins and their receptors in control and pre-eclamptic placentae and the responses of the TAC3 gene encoding NKB to proposed physiological triggers of pre-eclampsia including hypoxia and oxidative stress using real-time quantitative PCR. We have determined the placenta to be the main site of TAC3 expression with levels 2.6-fold higher than the brain. TAC3 expression was found to be significantly higher in pre-eclamptic placenta (1.7-fold, P < 0.05) than in normal controls. No evidence was found that hypoxia and oxidative stress were responsible for increases in TAC3 expression. In rat placenta, a longitudinal study in normal late pregnancy was associated with a significant down-regulation of the NKB/NK3 ligand-receptor pair (P < 0.05). The present data suggest that the increased placental expression of TAC3 is part of the mechanism leading to the increased circulating levels of NKB in pre-eclampsia.

Hemokinins and endokinins.

The mammalian tachykinins are a family of peptides that, until recently, has included substance P (SP), neurokinin A and neurokinin B. Since, the discovery of a third preprotachykinin gene ( TAC4), the number of tachykinins has more than doubled to reveal several species-divergent peptides. This group includes hemokinin-1 (HK-1) in mouse and rat, endokinin-1 (EK-1) in rabbit, and EKA, EKB, human HK-1 (hHK-1) and hHK(4-11) in humans. Each exhibits a remarkable selectivity and potency for the tachykinin NK(1) receptor similar to SP. Their peripheral expression has led to the proposal that they are the endogenous peripheral SP-like endocrine/paracrine agonists where SP is not expressed. Moreover, their strong cross-reactivity with a specific SP antibody leads us to question many of the proposed locations and roles of SP in the periphery. Additionally, three orphan tachykinin gene-related peptides are identified on TAC4, in rabbit, EK-2 and in humans, EKC and EKD.

Neurokinin B is a paracrine vasodilator in the human fetal placental circulation.

Neurokinin (NK) B is a member of the tachykinin family of neurotransmitters, exerting hypotensive or hypertensive effects in the mammalian vasculature through synaptic release from peripheral neurons, according to either NK(1) and NK(2) or NK(3) receptor subtype expression, respectively. There is recent evidence that NKB is expressed by the syncytiotrophoblast of the human placenta, an organ that is not innervated. We hypothesized that NKB is a paracrine modulator of tone in the fetal placental circulation. We tested this hypothesis using the in vitro perfused human placental cotyledon. Our data show that NKB is a dilator of the fetal vasculature, causing a maximal 25.1 +/- 4.5% (mean +/- SEM; n = 5) decrease in fetal-side arterial hydrostatic pressure (5- microM NKB bolus; effective concentration in the circulation, 1.89 nM) after preconstriction with U-46619. RT-PCR demonstrated the presence of mRNA for NK(1) and NK(2) tachykinin receptors in the placenta. Using selective receptor antagonists, we found that NKB-induced vasodilation is through the NK(1) receptor subtype. We found no evidence for the involvement of either nitric oxide or prostacyclin in this response. This study demonstrates a paracrine role for NKB in the regulation of fetal placental vascular tone.

The characterization of pregnancy associated plasma protein-E and the identification of an alternative splice variant.

We have performed differential display and bioinformatic database mining of the placenta, in an attempt to find novel diagnostic markers of pathological pregnancies. We have identified a full-length cDNA encoding the preproprotein of pregnancy associated plasma protein-E (PAPP-E); a putative metalloprotease, of 1790-residues with a putative 21-residue signal peptide. An alternatively spliced mRNA was found to encode an 826-residue precursor protein corresponding to the N-terminus of PAPP-E. Both PAPP-E variants were found to be co-expressed abundantly in the placenta and non-pregnant mammary gland with low expression in the kidney, foetal brain and pancreas. Analysis of the predicted proteins suggests that the longer variant be targeted to the nucleus while the shorter variant is secreted extracellularly. Gene structure analysis revealed that PAPP-E was encoded on 23 exons on chromosome 1 and its splice variant on the first five same exons. The discovery of the PAPP-E variants will help in the deciphering of the physiology of this new family of metzincins in not only the placenta during pregnancy but also the mammary gland in breast cancer. The new PAPP-E variants could have the potential for the diagnosis of pathological pregnancies including trisomies such as Down's syndrome.

The role of neurokinin B in pre-eclampsia.

Pre-eclampsia, a life-threatening disease unique to pregnancy, has been called a disease of theories. To date, there has been no widely accepted predictive test or therapeutic intervention to prevent or delay pre-eclampsia. The discovery of a new placental hormone, neurokinin B, may finally help to answer some of the past mysteries.

The human apolipoprotein L gene cluster: identification, classification, and sites of distribution.

We report the cloning of a new gene family encoding six apolipoprotein L (apoL-I to -VI) proteins. The genes were identified as a cluster spanning a region of 619 kb on chromosome 22. Each apoL was found to share significant identity in its predicted amphipathic alpha helices while phylogenetic tree mapping showed the genes to be evolutionarily conserved. Tissue distribution by semiquantitative PCR revealed expression in all tissues, but consistently higher levels in the placenta were observed, except for apoL-V, which had a restricted expression. A comparison of tissue distribution with apoA-I, the major structural component of high-density lipoprotein, suggests that the apoL proteins may play a general and fundamental role in lipid biochemistry. In situ hybridization for expression of apoL-I in the placenta revealed expression throughout this tissue. The pathological expression of the apolipoproteins during pregnancy is implicated in fetal growth retardation, preeclampsia, and the onset of adult atherosclerosis.

A regulatory role for neurokinin B in placental physiology and pre-eclampsia.

Tachykinin dogma has assumed, so far, that neurokinin B (NKB) is a neuropeptide that is not produced in any peripheral tissue even though its endogenous receptor, NK3, has been found in a number of locations throughout the human body. We have found an abundant source of peripheral NKB in the human and rat placenta. In this review we describe the discovery of NKB in the placenta and examine its possible role in placental physiology and pre-eclampsia (PE). Excessive secretion of placental NKB into the maternal circulation during the third trimester of pregnancy has been found in women suffering from PE. This may provide the key to the cause of the multiple and complex symptoms associated with this potentially life-threatening illness. We also reveal the structural organisation of the human NKB gene for the first time as well as discussing putative mechanisms for its control.

Excessive placental secretion of neurokinin B during the third trimester causes pre-eclampsia.

Pre-eclampsia is a principal cause of maternal morbidity and mortality, affecting 5-10% of first pregnancies worldwide. Manifestations include increased blood pressure, proteinuria, coagulopathy and peripheral and cerebral oedema. Although the aetiology and pathogenesis remain to be elucidated, the placenta is undoubtedly involved, as termination of pregnancy eradicates the disease. Here we have cloned a complementary DNA from human placental messenger RNA encoding a precursor protein of 121 amino acids which gives rise to a mature peptide identical to the neuropeptide neurokinin B (NKB) of other mammalian species. In female rats, concentrations of NKB several-fold above that of an animal 20 days into pregnancy caused substantial pressor activity. In human pregnancy, the expression of NKB was confined to the outer syncytiotrophoblast of the placenta, significant concentrations of NKB could be detected in plasma as early as week 9, and plasma concentrations of NKB were grossly elevated in pregnancy-induced hypertension and pre-eclampsia. We conclude that elevated levels of NKB in early pregnancy may be an indicator of hypertension and pre-eclampsia, and that treatment with certain neurokinin receptor antagonists may be useful in alleviating the symptoms.

Induction of a serotonergic and neuronal phenotype in thyroid C-cells.

We have investigated whether rat thyroid C-cells can acquire a phenotype similar to serotonergic neurons. C-cells are neural crest derived endocrine cells with some intrinsic neuronal and serotonergic properties. A relatively simple isolation scheme yielded cultures of about 50% initial purity, as measured by fluorescence activated cell sorting. These enriched C-cells could extend neurites up to 550 microns on a laminin-containing substratum in the presence of NGF. The cultured C-cells expressed neurofilaments and this expression was enhanced by NGF treatment. The C-cells also expressed two markers of the sympathoadrenal neural crest lineage, the mammalian achaete scute homolog-1 (MASH-1) transcription factor, and the B2 cell surface antigen. Interestingly, MASH-1 was not detectable after the C-cells were placed in culture, which is consistent with neuronal differentiation, since MASH-1 is only expressed in neuronal progenitors prior to differentiation. We then demonstrated that C-cells possess the fundamental features of serotonergic neurons: synthesis and secretion, uptake, and feedback control. The enriched C-cells, as well as the CA77 C-cell line, showed 5-HT immunostaining, expression of tryptophan hydroxylase mRNA, 5-HT1B autoreceptor mRNA, and 5-HT transporter mRNA and activity. NGF greatly induced 5-HT transporter activity as determined by sensitivity to sertraline, a selective 5-HT reuptake inhibitor. Based on these results, we propose that thyroid C-cells are derived from a vagal sympathoadrenal progenitor, similar to serotonergic enteric neurons, and can undergo neuronal transdifferentiation. Hence, these cells should provide suitable and convenient models for molecular and cellular studies on serotonergic neurons.