CCN1 (CYR61) and CCN3 (NOV) signaling drives human trophoblast cells into senescence and stimulates migration properties

ABSTRACT

During placental development, continuous invasion of trophoblasts into the maternal compartment depends on the support of proliferating extravillous trophoblasts (EVTs). Unlike tumor cells, EVTs escape from the cell cycle before invasion into the decidua and spiral arteries. This study focused on the regulation properties of glycosylated and non-glycosylated matricellular CCN1 and CCN3, primarily for proliferation control in the benign SGHPL-5 trophoblast cell line, which originates from the first-trimester placenta. Treating SGHPL-5 trophoblast cells with the glycosylated forms of recombinant CCN1 and CCN3 decreased cell proliferation by bringing about G0/G1 cell cycle arrest, which was accompanied by the upregulation of activated Notch-1 and its target gene p21. Interestingly, both CCN proteins increased senescence-associated β-galactosidase activity and the expression of the senescence marker p16. The migration capability of SGHPL-5 cells was mostly enhanced in response to CCN1 and CCN3, by the activation of FAK and Akt kinase but not by the activation of ERK1/2. In summary, both CCN proteins play a key role in regulating trophoblast cell differentiation by inducing senescence and enhancing migration properties. Reduced levels of CCN1 and CCN3, as found in early-onset preeclampsia, could contribute to a shift from invasive to proliferative EVTs and may explain their shallow invasion properties in this disease.     

External anions relieve DIDS inhibition of SO4 efflux from placental membrane vesicles

Sulphate efflux from human placental microvillus membrane vesicles was inhibited by external DIDS (K_I10 .6 M). This inhibition was partially reversed on addition of the translocated substrates sulphate or selenate to the external medium: selenite which is not translocated does not protect against DIDS inhibition. These findings show that the mechanism responsible for sulphate efflux can be modified by substrate in the external medium.Abbreviations DIDS 4-acetamido-4-isothiocyanostilbene-2-2-disulphonate     

Adenosine transport and nitrobenzylthioinosine binding in human placental membrane vesicles from brush-border and basal sides of the trophoblast

Summary The nucleoside transport activity of human placental syncytiotrophoblast brush-border and basal membrane vesicles was compared. Adenosine and uridine were taken up into an osmotically active space. Adenosine was rapidly metabolized to inosine, metabolism was blocked by preincubating vesicles with 2-deoxycoformycin, and subsequent adenosine uptake studies were performed in the presence of 2-deoxycoformycin. Adenosine influx by brush-border membrane vesicles was fitted to a two-component system consisting of a saturable system with apparent Michaelis-Menten kinetics (apparentK _m approx. 150 m) and a linear component. Adenosine uptake by the saturable system was blocked by nitrobenzylthioinosine (NBMPR), dilazep, dipyridamole and other nucleosides. Inhibition by NBMPR was associated with high-affinity binding of NBMPR to the brush-border membrane vesicles (apparentK _d 0.98±0.21nm). Binding of NBMPR to these sites was blocked by adenosine, inosine, uridine, thymidine, dilazep and dipyridamole, and the respective apparentK _i values were 0.23±0.012, 0.36±0.035, 0.78±0.1, 0.70±0.12 (mm), and 0.12 and 4.2±1.4 (nm). In contrast, adenosine influx by basal membrane vesicles was low (less than 10% of the rate observed with brush-border membrane vesicles under similar conditions), and hence no quantitative studies of adenosine uptake could be performed with these vesicles. Nevertheless, high-affinity NBMPR binding sites were demonstrated in basal membrane vesicles with similar properties to those in brushborder membrane vesicles (apparentK _d 1.05±0.13nM and apparentK _i values for adenosine, inosine, uridine, thymidine, dilazep and dipyridamole of 0.14±0.045, 0.54±0.046, 1.26±0.20, 1.09±0.18mm and 0.14 and 3.7±0.5nm, respectively). Exposure of both membrane vesicles to UV light in the presence of [³H]NBMPR resulted in covalent labeling of a membrane protein(s) with a broad apparentM _r on SDS gel electropherograms of 77,000–45,000, similar to that previously reported for many other tissues, including human erythrocytes. We conclude that the maternal (brush-border) and fetal (basal) surface of the human placental syncytiotrophoblast posses broad-specificity, facilitated-diffusion, NBMPR-sensitive nucleoside transporters.     

Gestational changes in electrolyte transport,electrical activity,and permeability of the porcine placenta

Summary 1. The electrolyte transport capacities of the porcine placenta and fetal membranes (amnion, chorion, and allantois) during gestation (47–112 days) were assessed in vitro and in the absence of electrochemical, osmotic, or hydrostatic driving forces. Net transmural transport of ²²Na and ³⁶Cl across sections of porcine allantochorion-endometrium (ALCE), amniochorionendometrium (AMCE), and allantoamnion (AA) mounted in Ussing chambers could not be detected at any stage of gestation. 2. These tissues were characterised throughout gestation by low electrical potential difference (PD; <5 mV, fetal side positive with respect to maternal side) and short circuit current (SCC; <16 A · cm^-1) which were amiloride and iodoacetate sensitive. 3. A periparturitent reversal in the polarity of PD to fetal side negative and a change in the direction of the SCC was observed in tissues obtained from sows (n=3) at 111–112 day gestation. These changes were accompanied by a 73% increase in electrical resistance (R) and a 2–10-fold decrease in the permeability of tissues to Na and Cl. 4. Morphological studies revealed a decrease in the thickness of allantoamnion and a decrease in the height of epithelia from ALCE, AMCE, and AA during gestation, indicating a decrease in activity. These morphological changes only partly explained the pre-term electrophysiological changes in these tissues.Abbreviations AA allantoamnion - ALCE allantochorionendometrium - AMCE Amniochorion-endometrium - fm feto-maternal - mf materno-fetal - PD electrical potential difference - PVP polyvinyl pyrrolidone - R resistance - SCC short circuit current     

Relationship between fatty acid binding proteins,acetyl-CoA formation and fatty acid synthesis in developing human placenta

The relationship between fatty acid binding proteins, ATP citrate lyase activity and fatty acid synthesis in developing human placenta has been studied. Fatty acid binding proteins reverse the inhibitory efect of palmitoyl-CoA and oleate on ATP citrate lyase and fatty acid synthesis. In the absence of these inhibitors fatty acid binding proteins activate ATP citrate lyase and stimulate [ 1-¹⁴ C] acetate incorporation into placental fatty acids indicating binding of endogenous inhibitors by these proteins. Thus these proteins regulate the supply of acetyl-CoA as well as the synthesis of fatty acids from that substrates. As gestation proceeds and more lipids are required by the developing placenta fatty acid binding protein content, activity of ATP citrate lyase and rate of fatty acid synthesis increase indicating a cause and efect relationship between the demand of lipids and supply of precursor fatty acids during human placental development.     

Stimulation of proliferation of bovine placental cells by products of activated mononuclear leukocytes

Summary Culture medium conditioned with concanavalin A-stimulated mononuclear leukocytes was tested for its ability to stimulate in vitro proliferation of bovine placental cells. The crude preparation of cytokines caused a dose-dependent increase in [³H]thymidine uptake into cells obtained by trypsinization of fresh bovine placentae and placental cell lines established from cellular outgrowths of long-term bovine placental cultures, but had no effect on growth of 3T3 fibroblasts. Growth of trypsinized placental cells was not enhanced by culture in the presence of interleukin-2, interferon-β₂, interferon-γ, or tumor necrosis factor-α. These results corroborate those of murine studies, suggesting a growth-promoting role for cytokines released into the maternal-fetal interface. Supported in part by a grant from th e Florida Dairy Farmers Milk Checkoff Fund and a grant from CIBA-GEIGY. This is Journal Series R-01079 of the Florida Agricultural Experiment Station.     

A study of selenate efflux from human placental microvillus membrane vesicles

Selenate efflux from human placental brush border membrane vesicles was studied using an ion-exchange column assay. Selenate efflux was found to be mediated almost exclusively by a temperature dependent DIDS-sensitive pathway. Chromate markedly inhibited selenate efflux: in contrast medium selenate had no effect. It is concluded that selenate and sulphate share a common pathway for transport across the human placental microvillus membrane.Abbreviations DIDS 4-acetamido-4-isothiocyanostilbene-2-2-disulphonate     

Prenatal effects of transplacental exposure to ifosfamide in rats

Ifosfamide is an alkylating chemotherapeutic agent that exhibits activity against a wide range of tumors. Exposure to such agent just prior to mating (preconception period) may have adverse effects on developing embryos. I investigated the rate of apoptosis and the histological changes in both placenta and developing fetal tissues after exposure to ifosfamide of young female rats before mating. I clarified the roles of the drug and the placenta in causing fetal developmental toxicity. Rats were divided into four groups: (1) untreated controls, (2) rats administered saline, (3 and 4) rats administered 25 mg/kg and 50 mg/kg ifosfamide, respectively. After treatment of females with ifosfamide, the treated females were allowed to mate with normal untreated males. All pregnant animals were sacrificed on day 18 of gestation. Treatment with high doses of ifosfamide caused small placentas, fewer viable fetuses, greater post-implantation losses and more resorbed fetuses. Reduced progesterone and increased prolactin levels also were found. Immunohistochemical staining, the TUNEL technique and histological studies showed increased apoptotic cells and many histological changes in the placenta, and in fetal brain, liver and kidney tissues. Ifosfamide treatment increased apoptosis and caused hypoplasia of placental basal and labyrinth zones, which resulted in pathological changes in developing fetal tissue.     

Placental implications for pregnancy complications in the chimpanzee (Pan troglodytes)

Placentas from 28 term chimpanzee pregnancies, including two sets of dichorionic-diamniotic twin-pregnancies, were examined and compared histopathologically with those obtained from 171 small-for-dates (SFD), 306 premature, and 77 pregnancy toxemic human infants. Term infant and placental weights for the chimpanzee were generally smaller than for any of the human categories studied. Macroscopically, the chimpanzee placentas showed a high frequency of extrachorialis, infarctions, intervillous thrombi, and marginal hemorrhages, pathologies frequently associated with pregnancy toxemia and abruptio placenta in the human being. Yet, ultrastructurally, the chimpanzee chorionic villi evidenced well-developed organella, syncytial microvilli, and chorionic capillaries, although villitis and inflammation of the membranes and cord were not infrequently seen. These findings suggest that chimpanzees may suffer the same obstetric complications seen in human pregnancies.     

Pre-eclampsia onset and SPARC: A possible involvement in placenta development

Pre-eclampsia (PE) is a multisystem disorder commonly diagnosed in the latter half of pregnancy and it is a leading cause of intrauterine fetal growth retardation (IUGR). The aim of this study was to investigate the localization and the role of SPARC, secreted protein acidic, and rich in cysteine, in PE and PE–IUGR placentas in comparison with normal placentas. SPARC was mainly expressed in the villous and extravillous cytotrophoblastic cells in first trimester, whereas in PE, PE–IUGR and at term placentas, SPARC immunostaining was visible in both cytotrophoblastic cells and syncytiotrophoblast. SPARC expression significantly decreased in normal placenta from first to third trimester and a further significant reduction was demonstrated in PE and PE–IUGR. The latter downregulation of SPARC depends on hypoxic condition as shown by in vitro models. In conclusion, SPARC can play a pivotal role in PE and PE–IUGR onset and it should be considered as a key molecule for future investigations in such pathologies.