Inhibition of human placental endothelial cell proliferation and angiogenesis by netrin-4.

INTRODUCTION: Netrin-4 is a secreted member of the laminin-related protein family, known to be involved in axonal guidance and endothelial cell survival, proliferation, and migration. We have recently reported the cellular localization of netrin-4 and its receptor neogenin in human first trimester and term placenta. A strong expression of netrin-4 was observed in trophoblast and in endothelial cells, suggesting a potential role of this protein in placental angiogenesis. In relation to human pregnancy, it has been reported that circulating netrin-4 were increased in fetal umbilical cord blood of intrauterine growth restriction IUGR compared to normal pregnancy suggesting an adverse effect of this protein on placental and fetal development. The aim of this study was to determine the role of netrin-4 in placental angiogenesis. METHODS: The effects of netrin-4 on proliferation, migration, tube-like organization, and spheroid sprouting of human placental microvascular endothelial cells (HPEC) were studied. RESULTS: We demonstrated that netrin-4 inhibits HPEC proliferation, tube-like formation, migration and spheroid sprouting, suggesting a direct role of netrin-4 in the regulation of intra-villus angiogenesis. DISCUSSION: This is the first report of an anti-angiogenic activity of netrin-4 in human placenta. This study brings new insights into netrin-4 roles in placental angiogenesis and suggests possible involvements of netrin-4 in angiogenesis-related pathologies such as IUGR.

Endocrine gland-derived endothelial growth factor (EG-VEGF) is a potential novel regulator of human parturition.

EG-VEGF is an angiogenic factor that we identified as a new placental growth factor during human pregnancy. EG-VEGF is also expressed in the mouse fetal membrane (FM) by the end of gestation, suggesting a local role for this protein in the mechanism of parturition. However, injection of EG-VEGF to gravid mice did not induce labor, suggesting a different role for EG-VEGF in parturition. Here, we searched for its role in the FM in relation to human parturition. Human pregnant sera and total FM, chorion, and amnion were collected during the second and third trimesters from preterm no labor, term no labor, and term labor patients. Primary human chorion trophoblast and FM explants cultures were also used. We demonstrate that circulating EG-VEGF increased toward term and significantly decreased at the time of labor. EG-VEGF production was higher in the FM compared to placentas matched for gestational age. Within the FM, the chorion was the main source of EG-VEGF. EG-VEGF receptors, PROKR1 and PROKR2, were differentially expressed within the FM with increased expression toward term and an abrupt decrease with the onset of labor. In chorion trophoblast and FM explants collected from nonlaboring patients, EG-VEGF decreased metalloproteinase-2 and -9 activities and increased PGDH (prostaglandin-metabolizing enzyme) expression. Altogether these data demonstrate that EG-VEGF is a new cytokine that acts locally to ensure FM protection in late pregnancy. Its fine contribution to the initiation of human labor is exhibited by the abrupt decrease in its levels as well as a reduction in its receptors.

Characterization of the adverse effects of nicotine on placental development: in vivo and in vitro studies.

In utero exposure to nicotine is associated with increased risk of numerous adverse fetal and neonatal outcomes, which suggests that it acts directly to affect placental development and the establishment of the fetomaternal circulation (FC). This study used both in vivo [Wistar rats treated with 1 mg/kg nicotine from 2 wk prior to mating until gestational day (GD) 15] and in vitro (RCHO-1 cell line; treated with 10(-9) to 10(-3)M nicotine) models to examine the effects of nicotine on these pathways. At GD 15, control and treated placentas were examined for the impact of nicotine on 1) trophoblast invasion, proliferation, and degree of hypoxia, 2) labyrinth vascularization, 3) expression of key genes of placental development, and 4) expression of placental angiogenic factors. The RCHO-1 cell line was used to determine the direct effects of nicotine on trophoblast differentiation. Our in vivo experiments show that nicotine inhibits trophoblast interstitial invasion, increases placental hypoxia, downregulates labyrinth vascularization as well as key transcription factors Hand1 and GCM1, and decreases local and circulating EG-VEGF, a key placental angiogenic factor. The in vitro experiments confirmed the inhibitory effects of nicotine on the trophoblast migration, invasion, and differentiation processes and demonstrated that those effects are most likely due to a dysregulation in the expression of nicotine receptors and a decrease in MMP9 activity. Taken together, these data suggest that adverse effects of maternal smoking on pregnancy outcome are due in part to direct and endocrine effects of nicotine on the main processes of placental development and establishment of FC.

[Potential role of the angiogenic factor "EG-VEGF" in gestational trophoblastic diseases]. / Rôle potentiel du facteur angiogénique «EG-VEGF¼ dans les maladies gestationnelles trophoblastiques.

Gestational trophoblastic disease (MGT) includes a wide spectrum of pathologies of the placenta, ranging from benign precancerous lesions, with gestational trophoblastic tumors. Metastases are the leading causes of death as a result of this tumor. They represent a major problem for obstetrics and for the public health system. To date, there is no predictor of the progression of molar pregnancies to gestational trophoblastic tumor (GTT). Only an unfavorable plasma hCG monitoring after evacuation of hydatidiform mole is used to diagnose a TTG. The causes of the development of this cancer are still poorly understood. Increasing data in the literature suggests a close association between the development of this tumor and poor placental vascularization during the first trimester of pregnancy. The development of the human placenta depends on a coordination between the trophoblast and endothelial cells. A disruption in the expression of angiogenic factors could contribute to uterine or extra-uterine tissue invasion by extravillous trophoblast, contributing to the development of TTG. This review sheds lights on the phenomenon of angiogenesis during normal and abnormal placentation, especially during the MGT and reports preliminary finding concerning, the variability of expression of "Endocrine Gland-Derived Vascular Endothelial Growth Factor" (EG-VEGF), a specific placental angiogenic factor, in normal and molar placentas, and the potential role of differentiated expressions of the main placental angiogenic factors in the scalability of hydatidiform moles towards a recovery or towards the development of gestational trophoblastic tumor. Deciphering the mechanisms by which the angiogenic factor influences these processes will help understand the pathophysiology of MGT and to create opportunities for early diagnosis and treatment of the latter.

Effects of rosiglitazone on ovarian function and fertility in animals with reduced fertility following fetal and neonatal exposure to nicotine.

We have previously shown that in utero nicotine exposure causes impaired fertility, follicle immaturity, and ovarian dysfunction in adult female rat offspring. These characteristics overtly resemble the clinical profile of polycystic ovarian syndrome (PCOS) and recent studies have shown that thiazolidinediones such as rosiglitazone improve fertility in women with PCOS but the mechanism is not well defined. Our goal was to examine whether rosiglitazone would (1) ameliorate the altered ovarian physiology that occurs following fetal and neonatal exposure to nicotine and (2) to examine whether this could be due to normalization of ovarian vascularization. At weaning, offspring of nicotine-exposed dams were given either vehicle (NV) or rosiglitazone (3 mg kg(-1) day(-1); NR). Offspring of saline-exposed dams received vehicle (SV). Tissues were collected when the female offspring reached 26 weeks of age. NV animals had reduced granulosa cell proliferation and increased ovarian cell apoptosis. Treatment with rosiglitazone increased proliferation, and decreased apoptosis, compared NV animals. NV animals had decreased ovarian vascularity relative to controls, whereas NR animals had an intermediate level of ovarian vessel density. Moreover, ovaries from NV animals had decreased levels of the pro-angiogenic growth factors vascular endothelial growth factor (VEGF) and endocrine gland-derived VEGF both of which were increased with rosiglitazone treatment. Rosiglitazone reversed some of the nicotine effects in the ovary and increased ovarian vascularization, follicle maturation and improved oocyte competence. Rosiglitazone may be an important treatment option for PCOS and the present study provides a potential mechanism by which rosiglitazone may have beneficial effects on fertility in these patients.

Placental expression of EG-VEGF and its receptors PKR1 (prokineticin receptor-1) and PKR2 throughout mouse gestation.

Compelling evidence indicates that vascular endothelial growth factor (VEGF) is an important mediator of placental angiogenesis and appears to be disregulated in pre-eclampsia (PE). Recently, we characterised the expression of EG-VEGF (endocrine gland-derived vascular endothelial growth factor), also known as prokineticin 1 (PK1) in human placenta during the first trimester of pregnancy and showed that this factor is likely to play an important role in human placentation. However, because it is impossible to prospectively study placentation in humans, it has been impossible to further characterise EG-VEGF expression throughout complete gestation and especially at critical gestational ages for PE development. In the present study, we used mouse placenta to further characterise EG-VEGF expression throughout gestation. We investigated the pattern of expression of EG-VEGF and its receptors, PKR1 and PKR2 at the mRNA and protein levels. Our results show that EG-VEGF and VEGF exhibit different patterns of expression and different localisations in the mouse placenta. EG-VEGF was mainly localised in the labyrinth whereas VEGF was mainly present in glycogen and giant cells. EG-VEGF mRNA and protein levels were highest before 10.5days post coitus (dpc) whereas those of VEGF showed stable expression throughout gestation. PKR1 protein was localised to the labyrinth layer and showed the same pattern of expression as EG-VEGF whereas PKR2 expression was maintained over 10.5dpc with both trophoblastic and endothelial cell localisations. Altogether these findings suggest that EG-VEGF may have a direct effect on both endothelial and trophoblastic cells and is likely to play an important role in mouse placentation.

Effects of cortisol and oestradiol on hepatic 11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor proteins in late-gestation sheep fetus.

In the late-gestation sheep, increased fetal plasma cortisol concentration and placental oestradiol (E(2)) output contribute to fetal organ maturation, in addition to the onset of parturition. Both cortisol and E(2) are believed to regulate the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which interconverts bioactive 11-hydroxy glucocorticoids and their inactive 11-keto metabolites. 11beta-HSD1, abundantly expressed in fetal liver, operates primarily as a reductase enzyme to produce bioactive cortisol and thus regulates local hepatic glucocorticoid concentrations. Cortisol acts through the glucocorticoid receptor (GR) present in the liver. In this study, we examined the effects of cortisol and E(2) on hepatic 11beta-HSD1 and GR in the liver of chronically catheterized sheep fetuses treated with saline (n=5), cortisol (1.35 mg/h; n=5), saline+4-hydroxyandrostendione, a P450 aromatase inhibitor (4-OHA; 1.44 mg/h; n=5), or cortisol+4-OHA (n=5). Cortisol infusion resulted in increased plasma concentrations of fetal cortisol and E(2); concurrent administration of 4-OHA attenuated the increase in plasma E(2) concentrations. Using immunohistochemistry, we showed that fetal hepatocytes expressed both 11beta-HSD1 and GR proteins. Cortisol treatment increased GR in both cytosol and nuclei of hepatocytes; concurrent administration of 4-OHA was associated with distinct nuclear GR staining. Western blot revealed that cortisol, in the absence of increased E(2) concentrations, significantly increased concentrations of 11beta-HSD1 (34 kDa) and GR (95 kDa) proteins. 11beta-HSD1 enzyme activity was measured in the liver microsomal fraction in the presence of [(3)H]cortisone (10(-)(6) M) or [(3)H]cortisol (10(-)(6) M) and NADPH (reductase activity) or NADP(+) (dehydrogenase activity) respectively. 11beta-HSD1 reductase activity was significantly greater in the presence of cortisol. In summary, we found that, in sheep during late gestation, cortisol increased both 11beta-HSD1 and GR in the fetal liver, and these effects were accentuated in the absence of increased E(2).

Understanding preterm labor.

Increased uterine contractility at term and preterm results from activation and then stimulation of the myometrium. Activation can be provoked by mechanical stretch of the uterus and by an endocrine pathway resulting from increased activity of the fetal hypothalamic-pituitary-adrenal (HPA) axis. In fetal sheep, increased cortisol output during pregnancy regulates prostaglandin H synthase type 2 (PGHS2) expression in the placenta in an estrogen-independent manner, resulting in increased levels of PGE2 in the fetal circulation. Later increases in maternal uterine expresssion of PGHS2 require elevations of estrogen and lead to increased concentrations of PGF2alpha in the maternal circulation. Thus, regulation of PGHS2 at term is differentially controlled in fetal (trophoblast) and maternal (uterine epithelium) tissue. This difference may reflect expression of the glucocorticoid receptor (GR), but not estrogen receptor (ER), in placental trophoblast cells. In women, cortisol also contributes to increased PG production in fetal tissues through upregulation of PGHS2 (amnion and chorion) and downregulation of 15-OH PG dehydrogenase (chorion trophoblasts). The effect of cortisol on chorion expression of PGDH reverses a tonic stimulatory effect of progesterone, potentially through a paracrine or autocrine action. We have interpreted this interaction as a reflection of "progesterone withdrawal" in the primate, in relation to birth. Other agents, such as proinflammatory cytokines, similarly upregulate PGHS2 and decrease expression of PGDH, indicating the presence of several mechanisms by which labor at term or preterm may be initiated. These different mechanisms need to be considered in the development of strategies for the detection and management of the patient in preterm labor.

Vasopressin potentiates mineralocorticoid selectivity by stimulating 11 beta hydroxysteroid deshydrogenase in rat collecting duct.

Arginine vasopressin (AVP) and corticosteroid hormones are involved in sodium reabsorption regulation in the renal collecting duct. Synergy between AVP and aldosterone has been well documented, although its mechanism remains unclear. Both aldosterone and glucocorticoid hormones bind to the mineralocorticoid receptor (MR), and mineralocorticoid selectivity depends on the MR-protecting enzyme 11 beta hydroxysteroid deshydrogenase (11-HSD), which metabolizes glucocorticoids into derivatives with low affinity for MR. We have investigated whether the activity of 11-HSD could be influenced by AVP and corticosteroid hormones. This study shows that in isolated rat renal collecting ducts, AVP increases 11-HSD catalytic activity. This effect is maximal at 10(-8) M AVP (a concentration clearly above the normal physiological range of AVP concentrations) and involves the V2 receptor pathway, while activation of protein kinase C or changes in intracellular calcium are ineffective. The stimulatory effect of AVP on 11-HSD is largely reduced after adrenalectomy, and is selectively restored by infusion of aldosterone, not glucocorticoids. We conclude that this synergy between AVP and aldosterone in controlling the activity of 11-HSD is likely to play a pivotal role in resetting mineralocorticoid selectivity, and hence sodium reabsorption capacities of the renal collecting duct.

Prerequisite for cardiac aldosterone action. Mineralocorticoid receptor and 11 beta-hydroxysteroid dehydrogenase in the human heart.

BACKGROUND: It has been proposed that aldosterone exerts direct effects on heart function, most notably on the development of myocardial fibrosis during ventricular hypertrophy in rat. Initial events in aldosterone action entail its binding to mineralocorticoid receptor (MR). Because MR displays similar affinities for aldosterone and glucocorticoids, the in vivo aldosterone selectivity of MR requires the presence of an enzyme, 11 beta-hydroxysteroid dehydrogenase (11-HSD), which metabolizes glucocorticoids into inactive derivatives. Although evidence exists for the presence of MR in rodent heart, no data are available for humans; moreover, the existence of cardiac 11-HSD is controversial. METHODS AND RESULTS: The heart samples used originated from tissue removed during cardiac surgery in nontransplant patients or from endocavitary biopsies done for the follow-up of heart transplantation. The expression of MR was examined at the mRNA and protein level by in situ hybridization with cRNA probes specific for human MR mRNA and by immunodetection with two specific anti-MR antibodies. 11-HSD catalytic activity was determined by measurement of the metabolic rate of tritiated corticosteroids by cardiac samples. In nontransplanted hearts, an in situ hybridization signal equivalent to that found in the whole kidney was present on cardiomyocytes. Specific immunolabeling of cardiomyocytes with anti-MR antibodies demonstrated the presence of the MR protein. Cardiac 11-HSD activity was detected (243 +/- 26 fmol.30 min-1.mg protein-1) and was dependent on the cofactor NAD, not NADP, suggesting that it corresponds to the form of the enzyme specifically responsible for MR protection. In transplanted hearts that presented severe alterations, MR immunodetection was weaker and irregular, with no specific hybridization signal. CONCLUSIONS: Our results demonstrate that MR is coexpressed with 11-HSD in human heart, which thus possesses the cellular machinery required for direct aldosterone action.