Obesity in pregnancy stimulates macrophage accumulation and inflammation in the placenta.

Obesity and pregnancy are associated with a combination of insulin resistance and inflammatory changes which exacerbate in combination. Based on the similarity between the inflammatory transcriptomes of adipose tissue and placenta, we hypothesized that the placenta develops exaggerated inflammation in response to obesity. The aim of this study was to characterize placental inflammatory mediators and macrophage accumulation in relation to peripheral inflammation in obesity. Placental macrophages and maternal peripheral blood mononuclear cells (PBMC) from 20 obese and 15 lean women were functionally and phenotypically characterized using immunohistochemistry, flow cytometry and expression for macrophage markers and inflammatory cytokines. The number of resident CD68+ and CD14+ cells was increased 2-3 fold in the placenta of obese as compared to lean women. The macrophage population was characterized by a marked phenotypic heterogeneity with complex subsets of CD14+, CD68+ and CD11b+ (mac-1) cells and by an increased expression of the pro-inflammatory cytokines IL-1, TNF-alpha, IL-6. Placental inflammation was associated with an activation of PBMC gene expression with an increase in the monocyte differentiation and maturation markers CD14 and CD68 in maternal but not fetal PBMC. The inflammatory changes were associated with higher plasma concentrations of C-reactive protein and IL-6 in obese compared to lean women. In conclusion, the chronic inflammation state of pre-gravid obesity is extending to in utero life with accumulation of a heterogeneous macrophage population and pro-inflammatory mediators in the placenta. The resulting inflammatory milieu in which the fetus develops may have critical consequences for short and long term programming of obesity.

Junctions and adhesion molecules in first trimester and term human placentas.

Placental tight and gap junctions and their adhesion molecules were studied by immunochemistry and electron microscopy in early and term placentas in order to clarify their pattern of expression during placental development. Early syncytio-cytotrophoblast contained tight junctions with occludin and gap junctions with connexins 40 and 43. At term, endothelial cells from arterioles had tight and gap junctions following each other. Occludin, claudins 3 and 5 were found at the paracellular clefts of endothelial cells together with connexins 32, 40 and 50. Stromal cells had mixed tight and gap junctions with connexins 32, 43, 50. Capillaries demonstrated interendothelial tight junctions with claudins 3 and 5, and small gap junctions. Taken together these observations showed that the numerous tight and gap junctions of the early placental syncytio-cytotrophoblast are observed in the foetal arterioles and capillaries in the term placenta. We conclude that the tightness of the placenta due to the junctions lying in the syncytio-cytotrophoblast in early pregnancy is maintained by the foetal endothelial layer in term pregnancy, with significant developmental changes of their transmembrane proteins.

Immunocytological evidence for hematopoiesis in the early human placenta.

Hematopoiesis has previously been observed in the human yolk sac, in placental villi and in the embryonic aorta. Here, our immunocytological study at 24 and 35 days showed packed erythroblasts in the placental vessels, mitotic figures and anti-Ki-67 reactions within these cells. Morphologically, the erythroblasts and vessels were similar to those found in the yolk sac during primitive hematopoiesis. In addition, numerous extravascular erythroblasts were found in the villous core. Positive reactions were obtained in erythroblasts using antibodies against glycophorin-A, GATA-2 and C-kit that characterize the hematopoietic cells. However, erythroblasts did not react with anti-CD34 and anti-CD45. In this respect, they differ from the hematopoietic cell clusters observed in the aorta of the human embryo. The staining for glycophorin-A was maintained in erythroblasts at 6-7 weeks and 12-14 weeks. Anti-GATA-2 reaction was decreased in erythroblasts and appeared in the perivillous cytotrophoblast. Anti-C-kit signal was detected in endothelial cells at 6-7 weeks and switched to stromal and perivascular cells at 12-14 weeks. By term, anti-GATA-2 staining was still present in the trophoblast and appeared in vessels while anti-C-kit was negative. For the leukocytes marker CD15, a staining was found in the endothelium at 35 days, 6-7 and 12-14 weeks and in leukocytes at term. CD45 antibody decorated the leukocytes at 12-14 weeks and at term. Erythroblasts undergo a primitive hematopoiesis in the early placental vessels that may be of value for the embryo in a period of low oxygen environment.

Separation of trophoblastic and vascular cell lineages and vascular maturation in early human placental development.

The trophoblast and vascular cell lineages have been studied by immunohistochemistry at 6 and 12-14 weeks of pregnancy. Perivillous and extravillous cytotrophoblasts were specifically stained by anti-cytokeratin 7 whereas endothelial cells were labelled by anti-CD34 at these two stages of pregnancy. Perivillous and extravillous cytotrophoblasts together with erythroblasts showed mitotic figures and anti-Ki67 positive nuclei at the 6th week. In the perivillous cytotrophoblast, the number of Ki67 positive nuclei decreased by 12-14 weeks and the staining was limited to the proximal extravillous trophoblast of cell islands. Some endothelial and perivascular cells were labelled with anti-Ki67 at 12-14 weeks. Erythroblasts did not stain at all at this stage. Endothelial cells bound lectin UEA1 and vessels exhibited a fluorescent signal after anti-myosin staining at 12-14 weeks. These data showed that the cytotrophoblast and endothelial cell lineages are not related from 6 to 12-14 weeks of pregnancy. Because mitotic figures or anti-Ki67 staining were not observed in endothelium or perivascular cells at the 6th week, it seems likely that the endothelial cells committed to vasculogenesis derived from stromal cells. By 12-14 weeks, endothelial cells had nearly achieved their maturation by acquiring 1-fucosyl-binding sites revealed by UEA1-lectin binding and insuring their own renewal by mitosis. The maturation of perivascular cells at 12-14 weeks was shown by the anti-sm-myosin staining. We conclude that placental vasculogenesis involves mesenchymal cells rather than trophoblast. The differenciation of vessels organizing from random plexi to a vascular arborescence may involve paracrine regulatory loops with the trophoblast.

Placental leptin receptor isoforms in normal and pathological pregnancies.

Alternate mRNA splicing of human leptin receptor generates four membrane isoforms with different C-terminal sequences. They differ by the length of their intracellular domain which include specific motifs crucial for the specificity of leptin signalling. As a step towards functional studies, we have characterized leptin receptors in human placenta from normal pregnancies and pregnancies associated with diabetes and pre-eclampsia. Leptin and leptin receptors were visualized by immunohistochemistry of placentas obtained from first and third trimester pregnancies. Antibodies against N and C-terminal epitopes showed signals in the apical membrane of the syncytiotrophoblast in early and term placental villi as well as in JAr and BeWo derived trophoblast cells. In addition, a distinct isoform recognized by its extracellular juxtamembrane epitope was exclusively localized in cytotrophoblast cells and likely stains the soluble receptor. At contrast with the transmembrane receptors, the expression of this isoform is increased in placentas of pre-eclamptic and diabetic women which synthesize more leptin than placenta from uncomplicated pregnancy. These data demonstrate that short and long transmembrane leptin receptors are expressed in the trophoblast and indicate that leptin synthetized within the placenta can act locally through both receptor isoforms. Being also accessible to leptin from maternal origin, these transmembrane receptors may signal differently in pregnancy with normal and increased leptin production. The co-localization of leptin and the soluble receptor isoform suggests that this isoform serves for modulating maternal free leptin levels through modification of leptin binding capacities.

Characterization of first trimester human fetal placental vessels using immunocytochemical markers.

Cell differentiation markers on placental villi from the first trimester of human pregnancy have been studied by indirect immunofluorescence. Fluorescence labelling with antibodies against CD34 and CD31 was conspicuous in the vascular cells. The vascular paracellular clefts were labelled by anti-cadherin-5. A few vascular cells exhibited a positive reaction for von Willebrand factor, high-molecular-weight melanoma-associated-antibody and alpha-sm-actin compared to term pregnancy, indicating changes in protein expression during vascular differentiation. The poor anti-collagen IV reaction and the absence of a sm-myosin fluorescent signal observed around the vessels confirned the immaturity of the vessels. In contrast, strong reactions have previously been obtained with the latter antibodies in similar locations using term placental villi. A labelling was observed for antibodies against alpha3 and alpha5 integrins in these immature placental vessels suggesting cell-matrix interactions with specific domains of laminin or fibronectin. The vascular cells were also stained by anti-CD26. Surprisingly, the fetal vascular cells exhibited immunostainings in common with the villous cytotrophoblast (CD26) or the syncytiotrophoblast (cadherin-5) and cell islands cytotrophoblast (CD31, cadherin-5, alpha3 and alpha5 integrin subunits). These observations suggested a two step process for fetal vasculogenesis in the villi: i/ the formation of peripheral vessels induced by growth factors or cytokines derived from the nearby trophoblast, ii/ the development of muscular vessels due to growth factors or cytokines production induced by circulatory changes.

Phenotype of cultured fetal perivascular cells from human placenta studied by scanning electron microscopy.

The phenotype of perivascular placental cells has previously been studied using tissue sections from the fetal villi. The examination of these cells in culture by scanning electron microscopy gives us the opportunity to observe their three-dimensional phenotypes and associations outside their normal constraints. Human umbilical endothelial cells, which have a phenotype comparable to that observed in other studies, seem more flattened in culture than in their usual environment. Microvascular endothelial cells did not attain an epithelioid phenotype with close contacts between cells but formed a network of branched, elongated cells with phagocytotic activity. Some circular associations were observed when using a gelatinized matrix. Microvascular pericytes were large, flattened cells with an irregular border that pushed up nodular associations on a gelatin matrix. Chorioplacental myocytes adopted a network template comparable to that developed by microvascular endothelial cells. However, these elongated cells were thicker, without microvilli, and superficial filaments could be observed. In culture, confluent endothelial cells from the umbilical cord or microvascular pericytes associated as nodules reached a cell phenotype close to their in vivo counter-parts. This attainment of an in vivo phenotype remains questionable for chorioplacental myocytes. Microvascular endothelial cells, however, though there was sparse formation of circular associations, remained far from their in vivo phenotype.

Passage of S(+) and R(-) gamma-vinyl-GABA across the human isolated perfused placenta.

1. The maternal to foetal transfers of S(+)- and R(-)-gamma-vinyl-GABA (VGB) across the human isolated perfused placenta were low and comparable with those of acidic alpha-amino acids. 2. The placental uptake of the active S(+)-isomer from the maternal circulation exceeded that of the R(-)-isomer and this was reflected by a corresponding difference in placental tissue concentrations. 3. During perfusion with recirculation of the foetal medium, the two enantiomers were present at a similar concentration and did not concentrate in foetal perfusate, indicating that the excess amount of S(+)-VGB cleared from the maternal circulation was not accessible to the foetal perfusate. Furthermore, stable concentrations of both isomers in the foetal perfusate suggested a lack of placental metabolism. 4. Possible explanations of these findings include the operation of a stereoselective sodium-dependent-GABA placental uptake system on the maternal side, similar to that observed in neuronal tissue, or stereoselective binding to a placental GABA transaminase.

[Placental passage of macromolecules: transport pathway and mode]. / Passage placentaire des macromolécules: voie et mode de transport.

In vivo and in vitro data have recently shown that macromolecules cross the hemochorial placenta. This feature contrats with the size selectivity of the sheep placenta which exclude molecules of 0.45 nm radius. Macromolecules moves by diffusion or endocytosis. In addition to size selectivity, the electrical charges of the macromolecules might be involved in placental transfer. The main resistance to placental transfer lie in the trophoblastic layer and the pathways of specific or unspecific transcytosis are not elicited. Membrane or fluid phase markers (peroxydases), carrier molecules (LDL, transferrin...), hormones and growth factors (insuline, EGF) are internalized by endocytosis into the trophoblast and further degraded or recycled. Immunoglobulins G, which are protected from degradation, reach the fetal circulation. The endothelial layer looks less selective and permits passage either by interendothelial diffusion or transcytosis.

[Activities evoked in different strain structures by stimulation of the contralateral tectum in the chick]. / Activités évoquées dans les différentes structures striares par stimulation du tectum contra-latéral chez le poussin.

In the chick, as soon as hatching, the responses recorded in various areas in the "accessorius hyperstriatum" (Wulst) seem to be very localized in relation with the stimulated region of the contralateral tectum. Very similar results are obtained within the other striate structures. These observations suggest in first part, that the tectal efferents project very diffusely to various striate structures and, in other part, that it exists in these a rather precise tectotopy and a columnar organisation. These results appear very constant during the first month of the hatching.

[Development of tectum-striatum and striatum-tectum connections in the chick]. / Evolution des relations tecto-striaires et strio-tectales chez le poussin

In the chick, polyphasic evoked responses displaying a first negative component have been registered in the superficial part of the "Wulst" after stimulation in the depth of the contra or ipsilateral optic tectum, as early as the first hours following hatching. Moreover, responses on the surface of the tectum have been elicited by contra or ipsilateral stimulation of the Wulst. Latencies of both responses decrease markedly with increasing age but, in the whole, ipsilateral responses remain more latent than contralateral responses.