Extraembryonic heparin-binding epidermal growth factor-like growth factor deficiency compromises placentation in mice.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) is expressed in the embryo and uterus at the implantation site, stimulating trophoblast invasive activity essential for placentation. The effect of extraembryonic HBEGF deficiency on placental development was investigated by breeding mice heterozygous for the Hbegf null mutation. On gestation day 13.5, the average placental weights of the wild-type (Hbegf+/+) and heterozygous (Hbegf+/-) mice were approximately 76 and 77 mg, respectively, as opposed to reduced average placental weights of approximately 61 mg in homozygous null (Hbgef-/-) females. In contrast, fetal weights were not significantly affected by genotype. HBEGF immunostaining in placental sections was Hbegf gene dosage-dependent, while expression of other EGF family members was comparable in Hbegf+/+ and Hbegf-/- placentas. Histological analysis revealed no apparent differences in trophoblast giant cells, but the spongiotrophoblast region was reduced compared to labyrinth (P < 0.05) in Hbegf null placentas. While no differences in cell apoptosis were noted, proliferation as assessed by nuclear Ki67 staining was elevated in the labyrinth and decreased in the spongiotrophoblast region of Hbegf-/- placentas. Labyrinth morphology appeared disrupted in Hbegf -/- placentas stained with laminin, a marker for capillary basement membrane, and the capillary density was reduced. Immunohistochemical staining revealed reduced vascular endothelial growth factor (VEGF) levels in both spongiotrophoblast and labyrinth (P < 0.01) regions of Hbegf-/- placentas. In vitro, HBEGF supplementation increases the expression of VEGF in a human trophoblast cell line. These findings suggest that trophoblast HBEGF promotes placental capillary formation by inducing VEGF in the developing placenta of mice.

Vascular apoptosis associated with meglumine antimoniate: In vivo investigation of a chick embryo model.

The vasculo-toxic effect of meglumine antimoniate (MA) was confirmed in our previous investigation. The current study investigates the association of this effect with altered VEGF-A and VEGF-R2 expression. Additional mechanisms by which MA causes vascular toxicity are not clearly understood. We hypothesized that MA may alter normal expression of apoptotic genes and cause vascular toxicity. The current investigation was designed to address this issue using a chick embryo model. Fertile chicken eggs were treated with MA and the extra-embryonic membrane (EEM) vasculature was evaluated by morphometric, molecular and immunohistochemistry assays. The results showed that MA not only altered apoptotic gene expression, but that this alteration may disturb the normal development of the vascular network and cause embryo malformation. The relative expression level of the CASP3, CASP7, CASP9, APAF1, AIF1 and TP53 genes increased in drug-exposed EEMs. In addition, IHC assay confirmed the low expression BCL2 and increased expression of Bax, which are associated with a high rate of apoptosis. We suggest that induction of an apoptotic signaling pathway can lead to vascular defects during embryo development and the consecutive cascade of events can lead to the embryo malformation.

FSH receptor (FSHR) expression in human extragonadal reproductive tissues and the developing placenta, and the impact of its deletion on pregnancy in mice.

Expression and function of the follicle-stimulating hormone receptor (FSHR) in females were long thought to be limited to the ovary. Here, however, we identify extragonadal FSHR in both the human female reproductive tract and the placenta, and test its physiological relevance in mice. We show that in nonpregnant women FSHR is present on: endothelial cells of blood vessels in the endometrium, myometrium, and cervix; endometrial glands of the proliferative and secretory endometrium; cervical glands and the cervical stroma; and (at low levels) stromal cells and muscle fibers of the myometrium. In pregnant women, placental FSHR was detected as early as 8-10 wk of gestation and continued through term. It was expressed on: endothelial cells in fetal portions of the placenta and the umbilical cord; epithelial cells of the amnion; decidualized cells surrounding the maternal arteries in the maternal decidua; and the stromal cells and muscle fibers of the myometrium, with particularly strong expression at term. These findings suggest that FSHR expression is upregulated during decidualization and upregulated in myometrium as a function of pregnancy. The presence of FSHR in the placental vasculature suggests a role in placental angiogenesis. Analysis of genetically modified mice in which Fshr is lacking in fetal portions of the placenta revealed adverse effects on fetoplacental development. Our data further demonstrate FSHB and CGA mRNAs in placenta and uterus, consistent with potential local sources of FSH. Collectively, our data suggest heretofore unappreciated roles of extragonadal FSHR in female reproductive physiology.

Lysophosphatidic acid produced by hen egg white lysophospholipase D induces vascular development on extraembryonic membranes.

Lysophosphatidic acid (lysoPtdOH), a lysophospholipid mediator, exerts diverse physiological effects, including angiogenesis, through its specific G-protein-coupled receptors. Previously, we showed that unfertilized hen egg white contains polyunsaturated fatty acid-rich lysoPtdOH and lysophospholipase D (lysoPLD). Here, we examined whether lysoPtdOH was produced by lysoPLD in the presence and absence of a hen fertilized ovum and what the physiological role of lysoPtdOH in hen egg white is. Mass spectrometry showed that fertilized hen egg white contained about 8 µM lysoPtdOH before incubation with an ovum, mainly comprised of 18:1- (12.6 %), 18:2- (37.8 %) and 20:4-molecular species (41.5 %). In an early gestation period, the lysoPtdOH was increased up to 9.6 µM, concomitant with a decrease in the level of polyunsaturated lysophosphatidylcholine (lysoPtdCho). Moreover, lysoPtdOH-degrading activities were found in egg white and the vitelline membrane, showing that these enzymes control lysoPtdOH levels in egg white. In an egg yolk angiogenesis assay, two lysoPtdOH receptor antagonists, Ki16425 and N-palmitoyl serine phosphoric acid (NASP), inhibited blood vessel formation induced by exogenously added 18:1-lysoPtdOH and its precursor lysoPtdCho on the hen yolk sac. Ki16425 and NASP also inhibited blood vessel formation in the chorioallantoic membrane (CAM). Furthermore, the relatively higher levels of LPA1, LPA2, LPA4 and LPA6 mRNA were present in the yolk sac and CAM. These results suggest that lysoPtdOH produced from lysoPtdCho by the action of lysoPLD in hen egg white is involved in the formation of blood vessel networks through several lysoPtdOH receptors on various extraembryonic membranes, including the yolk sac membrane and CAM.

Completely infarcted smooth muscle tumor of the placental membrane.

Non-trophoblastic tumor of the placenta is rare, and so is placental smooth muscle tumor. We report leiomyoma of the placental membrane, which was discovered on cesarean section. Histologically, the tumor was a benign leiomyoma with complete necrosis, and this finding was confirmed immunohistochemically. Only six cases of smooth muscle tumors of the placenta have been reported to date. This is the third report of leiomyoma involving the placental membrane.

Development of bovine embryos derived from reproductive techniques.

Assisted reproduction techniques have improved agricultural breeding in the bovine. However, important development steps may differ from the situation in vivo and there is a high mortality rate during the first trimester of gestation. To better understand these events, we investigated the development of embryos and fetal membranes following fixed-time AI (FTAI), IVF and nuclear transfer (NT). The onset of yolk-sac development was not normal in cloned embryos. Later steps differed from conditions in vivo in all three groups; the yolk-sac was yellowish and juxtaposed with the amniotic membrane. Vascularisation of the chorioallantoic membrane was relatively late and low in NT gestations, but normal in the others. The overall development of the embryos was normal, as indicated by morphology and regression analysis of growth rate. However, NT conceptuses were significantly smaller, with the livers in some embryos occupying the abdominal cavity and others exhibiting heart abnormalities. In conclusion, the yolk-sac and the cardiovascular system seem to be vulnerable to morphogenetic alterations. Future studies will focus on gene expression and early vascularisation processes to investigate whether these changes may be responsible for the high incidence of intrauterine mortality, especially in clones.

The effects of homocysteine and folic acid on angiogenesis and VEGF expression during chicken vascular development.

Homocysteine (Hcy) has been implicated in the development of cardiovascular developmental defects. Additionally, in experimental studies, vasculotoxic properties of Hcy have been described. Although Hcy has been identified as a vascular pathogen, little is known about the direct effects Hcy exerts during early embryonic vascular development. Angiogenesis is a critical process involved in embryo survival and development. There are limited studies on the effects of Hcy on early embryonic vasculogenesis and angiogenesis. Folic acid (FA) is a B vitamin essential in embryo development, and FA supplementation may lead to reduced Hcy levels. Therefore, the purpose of our study was to explore the effects of Hcy and FA on early embryonic vascular development. Embryonic day (E) 3.5 chicken embryos were treated with a sham, Hcy or FA solution. We developed a computational program for systematic analysis of microscopic images obtained from the extra embryonic vascular beds. These results were combined with real-time PCR data on the expression of VEGF-A and its receptor in these vascular beds. Our data show that Hcy exposure inhibits early vascular development, displayed by a significant reduction of vessel area and altered composition of the vascular beds. Vascular beds of Hcy embryos for the greater part consisted of vessels of the smallest diameters, compared to middle size vessels in control and FA embryos. Hcy also reduced expression of VEGF-A and VEGFR-2. No significant effects of FA were found. We conclude that Hcy exposure causes impaired early extra embryonic vascular development, shown by altered composition of the vascular beds as well as reduced expression of VEGF-A and VEGFR-2. These effects of Hcy, and the consecutive cascade of events, may be involved in the development of cardiovascular developmental defects.

Variation in macrophage migration inhibitory factor [MIF] immunoreactivity during bovine gestation.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in several aspects of the immune response. MIF appears to play important roles in materno-fetal immuno-tolerance during placental establishment, modulation and growth as studied in epitheliochorial porcine and hemochorial human and mouse placentae. Here we studied the bovine placenta being multiplex, villous and synepitheliochorial with a low degree of invasion, to see if MIF could be involved. Placental tissues sampled from 12 cows at 9 stages of gestation (days 18-250), and endometrial tissues from two non-pregnant animals were processed for immunohistochemistry. Bovine MIF was detected by Western blot using anti-human MIF monoclonal antibodies. An immunoreactive band of approximately 12kDa confirmed similarities between bovine and human MIFs. Compared to the non-pregnant stage with very faint staining, the caruncular epithelium during pregnancy showed stronger staining for MIF. The intercaruncular epithelium in non-pregnant endometrium showed some reaction apically with increasing intensity at uterine gland openings; in contrast, at day 18 of gestation this staining was markedly increased. During gestation both caruncular and trophoblast epithelium of the placentomes were positive with different intensity in relation to the gestational stage. In the uterine glands, some strongly stained cells were present. The mature binucleated trophoblast giant cells were negative throughout pregnancy. During reestablishment of vascularisation, the vasculature in the caruncular area showed MIF reactivity. While supporting involvement of MIF in different placental types, the spatio-temporal variation in the bovine placenta suggests a regulatory role for MIF mainly in the interhemal barrier and during vascular development.

Notch signaling regulates remodeling and vessel diameter in the extraembryonic yolk sac.

BACKGROUND: The signaling cascades that direct the morphological differentiation of the vascular system during early embryogenesis are not well defined. Several signaling pathways, including Notch and VEGF signaling, are critical for the formation of the vasculature in the mouse. To further understand the role of Notch signaling during endothelial differentiation and the genes regulated by this pathway, both loss-of-function and gain-of-function approaches were analyzed in vivo. RESULTS: Conditional transgenic models were used to expand and ablate Notch signaling in the early embryonic endothelium. Embryos with activated Notch1 signaling in the vasculature displayed a variety of defects, and died soon after E10.5. Most notably, the extraembryonic vasculature of the yolk sac displayed remodeling differentiation defects, with greatly enlarged lumens. These phenotypes were distinct from endothelial loss-of-function of RBPJ, a transcriptional regulator of Notch activity. Gene expression analysis of RNA isolated from the yolk sac endothelia of transgenic embryos indicated aberrant expression in a variety of genes in these models. In particular, a variety of secreted factors, including VEGF and TGF-ß family members, displayed coordinate expression defects in the loss-of-function and gain-of-function models. CONCLUSIONS: Morphological analyses of the in vivo models confirm and expand the understanding of Notch signaling in directing endothelial development, specifically in the regulation of vessel diameter in the intra- and extraembryonic vasculature. Expression analysis of these in vivo models suggests that the vascular differentiation defects may be due to the regulation of key genes through the Notch-RBPJ signaling axis. A number of these genes regulated by Notch signaling encode secreted factors, suggesting that Notch signaling may mediate remodeling and vessel diameter in the extraembryonic yolk sac via autocrine and paracrine cell communication. We propose a role for Notch signaling in elaborating the microenvironment of the nascent arteriole, suggesting novel regulatory connections between Notch signaling and other signaling pathways during endothelial differentiation.

Intravascular pillars and pruning in the extraembryonic vessels of chick embryos.

To investigate the local mechanical forces associated with intravascular pillars and vessel pruning, we studied the conducting vessels in the extraembryonic circulation of the chick embryo. During the development days 13-17, intravascular pillars and blood flow parameters were identified using fluorescent vascular tracers and digital time-series video reconstructions. The geometry of selected vessels was confirmed by corrosion casting and scanning electron microscopy. Computational simulations of pruning vessels suggested that serial pillars form along pre-existing velocity streamlines; blood pressure demonstrated no obvious spatial relationship with the intravascular pillars. Modeling a Reynolds number of 0.03 produced 4 pillars at approximately 20-µm intervals matching the observed periodicity. In contrast, a Reynolds number of 0.06 produced only 2 pillars at approximately 63-µm intervals. Our modeling data indicated that the combination of wall shear stress and gradient of shear predicted the location, direction, and periodicity of developing pillars.

Novel HLA-G-binding leukocyte immunoglobulin-like receptor (LILR) expression patterns in human placentas and umbilical cords.

Human placentas are sources of cytokines, hormones and other substances that program receptive cells. One of these substances is HLA-G, which influences the functioning of both leukocytes and endothelial cells. In this study, we investigated the possibility that these and/or other types of cells in extraembryonic fetal tissues might respond to HLA-G by interacting with one or another of the leukocyte immunoglobulin-like receptors (LILR). LILRB1 is expressed by most leukocytes and LILRB2 is expressed primarily by monocytes, macrophages and dendritic cells. Analysis of term placentas by immunohistochemistry and Real Time PCR demonstrated that LILRB1 and LILRB2 protein and specific messages are produced in the mesenchyme of term villous placenta but are differently localized. LILRB1 was abundant in stromal cells and LILRB2 was prominent perivascularly. Neither receptor was identified in trophoblast. Further investigation using double label immunofluorescence indicated that placental vascular smooth muscle but not endothelia exhibit LILRB2. Term umbilical cord exhibited the same LILRB2 patterns as term placenta. Samples obtained by laser capture dissection of vascular smooth muscle in umbilical cords demonstrated LILRB2 mRNA, and double label immunofluorescence showed that cord vascular smooth muscle but not endothelium exhibited LILRB2 protein. The presence of LILRB1 in placental stromal cells and LILRB2 in vascular smooth muscle strongly suggest that HLA-G has novel functions in these tissues that could include regulation of placental immunity as well as development and function of the extraembryonic vasculature.

Placentation in the Amazonian manatee (Trichechus inunguis).

Evidence from several sources supports a close phylogenetic relationship between elephants and sirenians. To explore whether this was reflected in similar placentation, we examined eight delivered placentae from the Amazonian manatee using light microscopy and immunohistochemistry. In addition, the fetal placental circulation was described by scanning electron microscopy of vessel casts. The manatee placenta was zonary and endotheliochorial, like that of the elephant. The interhaemal barrier comprised maternal endothelium, cytotrophoblasts and fetal endothelium. We found columnar trophoblast beneath the chorionic plate and lining lacunae in this region, but there was no trace in the term placenta of haemophagous activity. The gross anatomy of the cord and fetal membranes was consistent with previous descriptions and included a four-chambered allantoic sac, as also found in the elephant and other afrotherians. Connective tissue septae descended from the chorionic plate and carried blood vessels to the labyrinth, where they gave rise to a dense capillary network. This appeared to drain into shorter vessels near the chorionic plate. The maternal vasculature could not be examined in the same detail, but maternal capillaries ran rather straight and roughly parallel to the fetal ones. Overall, there is a close resemblance in placentation between the manatee and the elephant.

Micro-PIV measurements of blood flow in extraembryonic blood vessels of chicken embryos.

The hemodynamic characteristics of blood flow are important in the diagnosis of circulatory diseases, since such diseases are related to wall shear stress of cardiovascular vessels. In chicken embryos at early stages of development, it is possible to directly visualize blood flow inside blood vessels. We therefore employed a micro-PIV technique to assess blood flow in extraembryonic venous and arterial blood vessels of chicken embryos, using red blood cells (RBCs) as tracers and obtaining flow images of RBCs using a high-speed CMOS camera. The mean velocity field showed non-Newtonian flow characteristics. The blood flow in two venous vessels merged smoothly into the Y-shaped downstream vein without any flow separation or secondary flow. Vorticity was high in the inner regions, where the radius of curvature varied greatly. A periodic variation of temporally resolved velocity signals, due to beating of the heart, was observed in arterial blood vessels. The pulsating frequency was obtained by fast Fourier transform analysis using the measured velocity data. The measurement technique used here was useful in analyzing the hemodynamic characteristics of in vivo blood flow in chicken embryos.

Generation of a conditional Ppap2b/Lpp3 null allele.

Lpp3, formerly known as Pap2b, is a lipid phosphohydrolase enzyme. Some of its substrates and products are lipids with potent biological and signaling activities such as phosphatidic acid, lysophosphatidic acid, sphingosine-1-phosphate, diacylglycerol, sphingosine, and ceramide. Lpp3 is dynamically expressed during development and is widely distributed in adult tissues. Targeted inactivation of Lpp3 gene (Ppap2b) in the mouse results in embryonic lethality because of defects in extraembryonic vascular development and gastrulation. To study the participation of Lpp3 later in development and in specific cell lineages we generated a conditional allele of Ppap2b. This was accomplished by flanking critical exons, responsible for its catalytic activity with loxP sites. A generalized Cre-mediated recombination of this allele yielded a phenotype fundamentally identical to our previously reported Ppap2b null allele.

Morphologic alterations in ovine placenta and fetal liver following induced severe placental insufficiency.

OBJECTIVES: Umbilical-placental embolization with microspheres has been used as a model of placental insufficiency and intrauterine growth restriction (IUGR). However, the effects of embolization on placental structure and organ morphology of the resulting IUGR fetus are relatively unexplored. In this study using ovine fetuses, we determined the location and distribution of microspheres within the placenta and explored the extent of placental and fetal organ morphologic changes induced by placental embolization. We hypothesized that microspheres administered into the umbilical circulation over 4 days would cause placental damage without significant morphologic alterations in fetal kidney or liver. METHODS: Eleven pregnant sheep at 118 +/- 1 (SE) days' gestation were studied. In six fetuses, embolization was induced by injections of 15-microm diameter microspheres on 4 successive days into the fetal descending aorta proximal to the umbilical arteries. Five fetuses served as time controls. RESULTS: In embolized fetuses, microspheres were detected in the placenta embedded in the fetal cytotrophoblastic layer or maternal parenchyma adjacent to villous cytotrophoblasts. Fetal cytotrophoblasts appeared normal except for loss of distinct separation between fetal and maternal cell layers. Microspheres were also detected in the fetal membranes within capillaries. The body weights of embolized fetuses were lower than controls, as were the body weight-normalized liver but not kidney weights. In the liver of the embolized fetuses, the number of hematopoietic cell clusters was markedly reduced, whereas the fetal kidneys appeared normal. CONCLUSIONS: We conclude that after 4 days of umbilical-placental embolization, microspheres were concentrated at the fetal villi proximal to the apical maternal-fetal interface and in the fetal membranes. There were noticeable morphologic changes in the embolized placentas, with no apparent gross damage to the placenta. The reduction in fetal liver weight and liver extramedullary hematopoietic cell abundance associated with embolization may predispose the fetus to alterations in liver function that could persist after birth.

The nucleoside derivative 5'-O-trityl-inosine (KIN59) suppresses thymidine phosphorylase-triggered angiogenesis via a noncompetitive mechanism of action.

Thymidine phosphorylase (TPase) catalyzes the reversible phosphorolysis of pyrimidine deoxynucleosides to 2-deoxy-d-ribose-1-phosphate and their respective pyrimidine bases. The enzymatic activity of TPase was found to be essential for its angiogenesis-stimulating properties. All of the previously described TPase inhibitors are either pyrimidine analogues that interact with the nucleoside-binding site of the enzyme or modified purine derivatives that mimic the pyrimidine structure and either compete with thymidine or act as a multisubstrate (competitive) inhibitor. We now describe the inhibitory activity of the purine riboside derivative KIN59 (5'-O-tritylinosine) against human and bacterial recombinant TPase and TPase-induced angiogenesis. In contrast to previously described TPase inhibitors, KIN59 does not compete with the pyrimidine nucleoside or the phosphate-binding site of the enzyme but noncompetitively inhibits TPase when thymidine or phosphate is used as the variable substrate. In addition, KIN59 was far more active than other TPase inhibitors, previously tested by us, against TPase-induced angiogenesis in the chorioallantoic membrane assay. The observed anti-angiogenic effect of KIN59 was not accompanied by inflammation or any visible toxicity. Inosine did not inhibit the enzymatic or angiogenic activity of the enzyme, indicating that the 5'-O-trityl group in KIN59 is essential for the observed effects. In contrast with current concepts, our data indicate that the angiogenic activity of TPase is not solely directed through its functional nucleoside and phosphate-binding sites. Other regulatory (allosteric) site(s) in TPase may play an important role in the mechanism of TPase-triggered angiogenesis stimulation and apoptosis inhibition. Identification of these site(s) is important to obtain a better insight into the molecular role of TPase in the progression of cancer and angiogenic diseases.

Vascular endothelial growth factor activation of intramembranous absorption: a critical pathway for amniotic fluid volume regulation.

OBJECTIVE: The purpose of this review is to propose a critical role for vascular endothelial growth factor (VEGF) in mediating the transfer of amniotic fluid from the amniotic compartment through the fetal membranes and fetal surface of the placenta into fetal blood. METHODS: Experimental findings in humans and animal models on the action of VEGF in mediating fluid transfer are reviewed and interpreted in order to postulate a proposed mechanism for VEGF regulation of amniotic fluid absorption through the fetal membranes and placenta. RESULTS: Recent scientific advances suggest that up-regulation of VEGF gene expression in the amnion and chorion is associated with increased transfer of amniotic fluid into fetal blood. The possible mechanisms of action for VEGF appear to involve regulation of intramembranous blood vessel proliferation and membrane transport via passive permeation as well as nonpassive transcytotic vesicular movement of fluid. CONCLUSION: Currently evolving concepts suggest that amniotic fluid volume is regulated through modulation of the rate of intramembranous absorption of amniotic fluid by both passive and nonpassive mechanisms. The permeability factor VEGF appears to be a critical regulator of amniotic fluid transport in the fetal membranes.

Engineered fibrin matrices for functional display of cell membrane-bound growth factor-like activities: study of angiogenic signaling by ephrin-B2.

With the rapid increase in approaches to pro- or anti-angiogenic therapy, new and effective methodologies for administration of cell-bound growth factors will be required. We sought to develop the natural hydrogel matrix fibrin as platform for extensive interactions and continuous signaling by the vascular morphogen ephrin-B2 that normally resides in the plasma membrane and requires multivalent presentation for ligation and activation of Eph receptors on apposing endothelial cell surfaces. Using fibrin and protein engineering technology to induce multivalent ligand presentation, a recombinant mutant ephrin-B2 receptor binding domain was covalently coupled to fibrin networks at variably high densities. The ability of fibrin-bound ephrin-B2 to act as ligand for endothelial cells was preserved, as demonstrated by a concomitant, dose-dependent increase of endothelial cell binding to engineered ephrin-B2-fibrin substrates in vitro. The therapeutic relevance of ephrin-B2-fibrin implant matrices was demonstrated by a local angiogenic response in the chick embryo chorioallontoic membrane evoked by the local and prolonged presentation of matrix-bound ephrin-B2 to tissue adjacing the implant. This new knowledge on biomimetic fibrin vehicles for precise local delivery of membrane-bound growth factor signals may help to elucidate specific biological growth factor function, and serve as starting point for development of new treatment strategies.