Elizabeth A. Linton

Shedding of syncytiotrophoblast microvilli into the maternal circulation in pre-eclamptic pregnancies

Objective To investigate whether syncytiotrophoblast microvilli (STBM) are shed into the maternal circulation in increased amounts in pre‐eclamptic pregnancies as a possible cause of maternal vascular endothelial dysfunction.

Cutaneous expression of corticotropin-releasing hormone (CRH), urocortin, and CRH receptors

Studies in mammalian skin have shown expression of the genes for corticotropin‐releasing hormone (CRH) and the related urocortin peptide, with subsequent production of the respective peptides. Recent molecular and biochemical analyses have further revealed the presence of CRH receptors (CRH‐Rs). These CRH‐Rs are functional, responding to CRH and urocortin peptides (exogenous or produced locally) through activation of receptor(s)‐mediated pathways to modify skin cell phenotype. Thus, when taken together with the previous findings of cutaneous expression of POMC and its receptors, these observations extend the range of regulatory elements of the hypo‐thalamic‐pituitary‐adrenal axis expressed in mammalian skin. Overall, the cutaneous CRH/POMC expression is highly reactive to common stressors such as immune cytokines, ultraviolet radiation, cutaneous pathology, or even the physiological changes associated with the hair cycle phase. Therefore, similar to its central analog, the local expression and action of CRH/POMC elements appear to be highly organized and entrained, representing general mechanism of cutaneous response to stressful stimuli. In such a CRH/ POMC system, the CRH‐Rs may be a central element.—Slominski, A., Wortsman, J., Pisarchik, A., Zbytek, B., Linton, E. A., Mazurkiewicz, J., Wei, E. T. Cutaneous expression of corticotropin‐releasing hormone (CRH), urocortin, and CRH receptors. FASEB J. 15, 1678–1693 (2001)

Circulating markers of oxidative stress are raised in normal pregnancy and pre‐eclampsia

Objective To determine whether circulating markers of oxidative stress are elevated in pre‐eclampsia when appropriate precautions are taken to prevent in vitro oxidation

Vitamins C and E Inhibit Apoptosis of Cultured Human Term Placenta Trophoblast

Preeclampsia can be lethal to both mother and baby. The prominent symptoms of this syndrome are hypertension, proteinuria and oedema, resulting from an exaggerated aseptic systemic inflammatory response, triggered by placental factors shed into the maternal circulation. Syncytiotrophoblast microparticles (STBM) are one possible factor, shed when the placenta is exposed to stressors such as hypoxia/reperfusion. These can disrupt mitochondria, triggering apoptosis and necrosis, placental pathologies which are increased in preeclampsia. We tested the effects of antioxidant vitamins C (50 microM) and E (50 microM) on trophoblast in culture, using term villous cytotrophoblast preparations. Following Percoll gradient centrifugation and MHC class I expressing cell depletion of placenta digests, syncytial fragments were removed using anti-placental alkaline phosphatase antibody. This yielded cytotrophoblasts of consistently high purity. EGF (10 ng/ml) stimulated syncytialisation and hCG and progesterone production. However, mitochondrial induced apoptosis (MIA) was evident 96h post-isolation, as mitochondrial membrane potential loss and caspase 9 and caspase 3 activation. ROCK-1 cleavage and syncytiotrophoblast particle shedding increased concurrently with apoptosis induction. Vitamins blocked MIA and syncytiotrophoblast particle shedding and significantly increased hCG (p<0.005) and progesterone (p<0.02) concentrations in culture supernatants, reflecting the increased survival rates. Although more cells survived in culture, syncytialisation rate (%) was significantly reduced (p<0.005). We conclude that vitamins C and E can significantly reduce mitochondrial damage generated following syncytialisation in vitro. However, further work is required to determine whether antioxidant vitamins interfere with normal fusion processes.

Heterogeneous Immunocytochemical Reactivities of oCRF-41-Like Material in the Human Hypothalamus, Pituitary and Gastrointestinal Tract

Tissue specimens of human hypothalami, pituitaries and gastrointestinal tract were studied with an indirect immunoperoxidase technique using 6 different rabbit ovine corticotrophin-releasing factor (oCRF-41) antisera. All these antisera detected oCRF-41-like immunoreactivity in parvocellular neurones of the paraventricular nucleus of the hypothalamus and their nerve terminals adjacent to portal capillaries in the infundibular stem and posterior pituitary. 1 antiserum recognised oCRF-41-like immunoreactivity in the growth hormone cells of the anterior pituitary. 3 other antisera recognised oCRF-41-like immunoreactivity in mucosal cells of the gastric antrum. Pre-treatment of the antisera with sauvagine did not affect the immunostaining of the hypothalamic and gastric cells, but quenched the immunostaining of growth hormone cells in the anterior pituitary. It is concluded that (1) in human hypothalami a CRF is synthesized that is structurally very similar to oCRF-41; (2) in growth hormone cells of the anterior pituitary oCRF-41-like immunoreactive material can be detected which shares antigenic determinants with sauvagine; (3) in mucosal cells of the gastric antrum oCRF-41-like immunoreactivity is present that is comparable but probably not identical to hypothalamic CRF and is not sauvagine-like.

Plasma urocortin levels in the diagnosis of ovarian endometriosis.

OBJECTIVE: Urocortin is a neuropeptide, member of the corticotropin-releasing hormone family, that is produced by the human endometrium. Ovarian endometrioma is a prevalent gynecologic disorder still lacking specific serum markers. In the present study we measured systemic levels of urocortin to assess the diagnostic performance of its determination in distinguishing endometriomas from other benign ovarian cysts. METHODS: Plasma urocortin was measured by radioimmunoassay in women with ovarian endometrioma (n=40) and in women with benign, nonendometriotic ovarian cysts (n=40). The diagnostic accuracy of urocortin measurement was evaluated by receiver operating characteristic curve and compared with the standard marker, CA 125. To support the local origin of the peptide, we also evaluated its localization in endometriomas by immunohistochemistry and its concentrations in cyst fluid and peritoneal fluid of 12 women with endometrioma. RESULTS: Plasma urocortin levels were twice as high in women with endometrioma (median 49 pg/mL, interquartile range 41–63 pg/mL) than in the control group (19 [15–23] pg/mL, P<.001) and significantly higher in the cystic content of endometriomas than in the peritoneal fluid and plasma (P<.05). The peptide was immunolocalized in endometrioma glands and stromal capillary vessels. Elevated plasma urocortin levels detected 88% of the cases of endometrioma with 90% specificity, whereas CA 125 detected only 65% of the cases with the same specificity. CONCLUSION: Plasma urocortin is increased in women with endometriomas, and its measurement may be useful for the differential diagnosis of endometrioma compared with other benign ovarian cysts. LEVEL OF EVIDENCE: II

Levels of Maternal Plasma Corticotropin-Releasing Factor and Urocortin During Labor

Objective: Corticotropin-releasing factor (CRF) is produced by the placenta and intrauterine tissues and secreted in increasing amounts from early to term pregnacy. In the presecnce of labor, a more incisive increase in CRF levels has been described, and women with preterm labor or hose destined to have premature delivery have higher midpregnancy CRF levels than hose who deliver at term. Urocortin is a 40-amino acid peptide belonging to the CRF family, expressed by human trophoblast and fetal membranes, which has the same biologic effects as CRF. Acting on the same CRF receptors, urocortin stimulates myometrial contractility and ACTH and prostaglandin release from cultured human placental cells. Because no data exist about urocortin levels in the maternal circultation at parturition, we investigated whether maternal plasma urocortin and CRF levels change according to cervical dilatation in healthy pregnant women at term labor. Methods: In a cross-sectional study of labor, a single maternal blood sample was collected from healthy pregnant women at term (n = 40); in a second longitudinal sudy, plama samples were collected longitudinally in a subset of patients (n = 8) throughout labor, according to a Bishop score evaluation. Results: Both maternal plasma CRF and urocortin levels were higher in labor than those previously reported during pregnancy, but they did not change significantly during the different stages of labor when evaluated longitudinally. Some patients showed a trend toward increasing levels, whereas others had variable concentrations. Conclusion: Neither CRF nor urocortin levels changed during the progression of spontaneous labor.

Serum from preeclamptic women induces vascular cell adhesion molecule-1 expression on human endothelial cells in vitro: A possible role of increased circulating levels of free fatty acids

OBJECTIVE The object was to determine whether serum from preeclamptic women induces expression of vascular cell adhesion molecule-1 on cultured endothelial cells. STUDY DESIGN Endothelial cells were incubated with medium containing 20% serum (volume/volume) either from women with preeclampsia (n = 15) or from women with normal pregnancies (n = 15) matched for maternal age, gestational age, and parity. A further matched set of samples (n = 10) was exposed to endothelial cells that had previously been incubated in the presence or absence of vitamin E (40 micromol/L final concentration). Free fatty acids were determined in each sample. A mixture of free fatty acids (linoleic, oleic, and palmitic acids, 1:1:1) was added to serum from control subjects in increasing concentrations (70-280 micromol/L final concentration) to emulate preeclamptic serum and the preparation was exposed to endothelial cells. In each experiment vascular cell adhesion molecule-1 expression was determined after 16 hours of exposure by an enzyme-linked immunosorbent assay technique performed on the cell monolayer. RESULTS Preeclamptic serum had higher levels of free fatty acids than did that of control subjects (0.71 mmol/L, 95% confidence level 0.5-0.93, vs 0.36 mmol/L, 95% confidence level 0.28-0.43). There was a statistically significant increase in vascular cell adhesion molecule-1 expression on the endothelial cells exposed to preeclamptic serum compared with those exposed to control serum (optical density 0.17 vs 0.11). Vitamin E reduced the vascular cell adhesion molecule-1 expression of endothelial cells exposed both to preeclamptic and to control serum samples in a nonspecific manner. Addition of free fatty acids to normal pregnancy serum to mimic the effect of preeclampsia resulted in increased expression of vascular cell adhesion molecule-1 on the cells. CONCLUSION Preeclamptic serum induces vascular cell adhesion molecule-1 expression on human endothelial cells in vitro, an effect also produced by fatty acids. The elevated level of free fatty acids in women with preeclampsia may contribute to increased vascular cell adhesion molecule expression in vivo.

Corticotrophin‐Releasing Hormone Receptor Type 1: Generation and Characterization of Polyclonal Antipeptide Antibodies and their Localization in Pituitary Cells and Cortical Neurones in vitro

Corticotrophin‐releasing hormone (CRH) is a 41 amino acid neuropeptide which plays a major role in regulating the endocrine response to stress. CRH acts by first binding to specific receptors on the plasma membrane of target cells. A CRH receptor from a human corticotroph adenoma and rat brain has recently been cloned (CRH‐R1). In this paper, we have chosen three different peptide sequences within the CRH‐R1 molecule which bear no similarity to other members of this receptor subfamily (or indeed any known protein) and which are likely to be exposed on the surface of the native protein, for antibody production. Some of these fragments produced anti‐peptide antibodies of good titre which cross‐reacted with the CRH‐R1 receptor expressed in transiently transfected COS‐7 cells and in tissue extracts from rat cerebellum, cortex, pituitary gland and human myometrium, both in Western blots and in liquid‐phase radioimmunoassay. We used immunofluorescence techniques to localize the CRH receptor in transiently transfected COS‐7 cells, primary cultures of rat anterior pituitary (AP) cells, the corticotroph‐tumour cells AtT20 D16–16 and cortical neurons in primary culture. Our results indicate IR‐CRH‐R1 receptors have a punctate distribution on the plasma membrane of AP cells and AtT20 D16–16 cells. Whilst in AP cells their appearance is a fine punctate pattern, in AtT20 cells, they appear as large patches which could account for receptor clusters. Within primary cortical neurons, their distribution does not appear to be polarized. Our results suggest that distribution of CRH‐R1 receptors within the different cell‐types investigated depends not only on the amino acid sequence but also on cellular factors.

High maternal and fetal plasma urocortin levels in pregnancies complicated by hypertension.

Objective We evaluated maternal and fetal plasma levels and placental mRNA expression of urocortin, a placental vasoactive neuropeptide, in singleton pregnancies (n = 70) complicated by hypertensive disorders classified as gestational hypertension (n = 36), pre-eclampsia (n = 19), and pre-eclampsia complicated by intrauterine growth restriction (PE/IUGR, n = 15), and in 70 healthy normotensive singleton pregnancies. Methods Plasma levels were assayed by radioimmunoassay, fetal biometry by ultrasound scans, utero-placental and fetal perfusion by Doppler velocimetry, and placental urocortin mRNA expression by quantitative real time reverse transcriptase-polymerase chain reaction. The main outcome measures were the correlation of urocortin concentrations with patterns of the utero-placental and fetal circulation, and the early prediction of a poor neonatal outcome such as the occurrence of perinatal death and intraventricular hemorrhage. Results Maternal and fetal urocortin levels were significantly (both P < 0.001) higher in gestational hypertension, pre-eclampsia and PE/IUGR women than in controls, and correlated with Doppler velocimetry patterns. Fetal concentrations were significantly (P < 0.0001) higher than and significantly (P < 0.0001) correlated to maternal levels. Placental mRNA expression did not change. Ten out of 140 newborns had a poor neonatal outcome, with an overall prevalence of 7.14% (pretest probability). Using the receiver operator characteristics curve analysis cut-off values, the probability of a poor neonatal outcome was 66.7% when urocortin was used, and was 0% if levels were unaltered. Conclusions Maternal and fetal urocortin levels are increased in hypertensive disorders of pregnancy. Since urocortin has vasoactive properties, the evidence of increased urocortin levels in hypertensive disorders may represent an adaptive fetal response.