Placenta as a selective barrier to cellular traffic

Transplacental passage of cells from mother to fetus during murine pregnancy was examined by using glucose phosphate isomerase (GPI) or fluorescein tagging as markers. Female mice of strains (BALB/cCr X C3H/HeJ)/F1 or (A/J X C57BL/6J)F1 (both Gpi-1a/b) were mated to the corresponding Gpi-la males (BALB/cCr or A/J, respectively). Cells of the liver, blood, and/or spleen in the offspring were typed at days 15, 16, 17, or 18 of gestation, the day of delivery, or 1 day postpartum. Only two of 172 Gpi-1a/a mice obtained from these matings showed evidence of maternal cell trafficking. Sensitivity of the assay was 1% Gpi-1a/a population. Fluorescein-labeled BALB/cCr peripheral red blood cells (RBC) or white blood cells (WBC) were injected i.v. into syngeneically mated BALB/cCr mothers on day 18. After 24 hr, the blood or liver of the neonates was formalin-fixed and examined in the fluorescence-activated cell sorter (FACS). Some RBC crossed the placenta, but WBC were usually not detected in fetal liver in significant numbers. This technique was very sensitive, and we estimated that no more than 225 WBC could enter the fetus via this route. Thus, we conclude that passage of significant numbers of maternal WBC into the fetus is rare, and perhaps this passage is related to a placental abnormality.     

Ontogeny of insulin receptors in the rat hemochorial placenta

Binding of 125I-insulin to rat placental membranes was time and protein concentration dependent, reversible, and specific. Unlabeled porcine insulin competed for 125I-insulin binding with an IC50 of 65 nM, while IGF-I was much less potent with an IC50 of 2.12 mM. Specific binding of 125I-insulin decreased during the second half of gestation from Days 11 to 19. Scatchard analysis of the binding data for membranes prepared from Gestation Days 11 and 19 yielded typical curvilinear plots which showed a marked decrease in the number of binding sites in late gestation placenta. Beginning on Day 14, insulin binding was characterized with isolated labyrinth and basal zone portions of the hemochorial placenta. There was no evidence for differences in Kd values or the number of binding sites in these two functionally distinct portions of the rat placenta. Crosslinking of 125I-insulin followed by SDS-PAGE showed a single protein with a molecular weight of 130,000 from placental tissues on Gestation Days 11 and 19 and confirmed a gestational decrease in the number of insulin receptors. In solubilized, lectin-purified preparations from placenta and liver membranes, insulin stimulated the phosphorylation of a Mr 95,000 protein. 32P-incorporation into this 95,000 protein was stimulated fivefold by insulin in Day 11 placenta receptor, whereas no detectable 32P-incorporation was found in Day 19 placenta. Thus, while the alpha- and beta-subunits of insulin receptors in mid and late gestation placenta have molecular weights which are similar to receptors in maternal liver, data indicate the presence of a functional difference in insulin-stimulated kinase activities.     

Comparative electron microscopy of chorio-allantoic placental barrier in some Indian Chiroptera

In the present study the comparative ultrastructure of the definitive chorio-allantoic placental barrier has been studied in considerable detail in six species of bats, representing six different families and both suborders of Chiroptera, by electron microscopy, and these species illustrate different kinds of interhaemal membranes met with among bats. The definitive chorio-allantoic placenta of Rousettus leschenaulti is haemodichorial, since the syncytiotrophoblast and cytotrophoblast layers are present to term. The fine structure of the placental barrier in the labyrinth of the definitive placenta of Rhinopoma hardwickei hardwickei is essentially endotheliomonochorial due to the presence of a single layer of cytotrophoblast and maternal endothelial cells. The placenta of Taphozous melanopogon, examined electron-microscopically in the present study, shows a thick maternal endothelium, a continuous interstitial membrane and the presence of a single layer of syncytiotrophoblast. The placenta of Megaderma comprises a typical endotheliochorial labyrinth and the presence of two layers of trophoblast. In Rhinolophus rouxi, the mature placenta during advanced pregnancy resembles that of Megaderma, its labyrinth containing large maternal capillaries with maternal endothelial cells and the two layers of trophoblast. Finally, the placental barrier of Hipposideros fulvus fulvus is haemodichorial due to the presence of two layers of trophoblast and the absence of maternal endothelial cells.     

Effect of different doses of ionizing radiation on the binding of prostaglandin E2 in mouse tissues

The effect of various radiation doses on membrane reception of prostaglandin E2(PGE2) of spleen, small intestine, brain, and liver cells of mice was studied in dynamics. Irradiation of the animals with doses producing bone marrow, intestinal and cerebral forms of radiation sickness was shown to change specific binding of PGE2 to cell membranes of both radiosensitive tissues and a relatively radioresistant organ, the liver.     

Human placental ferritin receptor

Brush-border membranes from human placenta were prepared and their purity was clarified by biochemical and morphological methods. Ferritin binding to these prepared membranes was examined using horse spleen 125I-apoferritin, and was found to be completed within 10 min at 37 degrees C and pH 7.5. The amount of ferritin bound to the membranes was found to be proportional to the amount of membrane added and saturable for a given amount of the membrane in the presence of excess ligand. The membranes exhibited specific ferritin binding with a Ka of 2.3 X 10(7) M-1 at pH 7.5. A competitive binding assay indicated that horse spleen 125I-apoferritin binding was inhibited by a 10-fold molar excess of horse spleen ferric ferritin and a 500-fold molar excess of human transferrin. These results suggest that human placental brush-border membranes have specific receptors for horse spleen apoferritin molecules.     

Characterization of antigenic sialoglycoprotein subunits of the placental brush border membranes: Comparison with liver and kidney membrane subunits by two-dimensional electrophoresis

The sialoglycoprotein subunits of human placental brush border membranes were labeled by sequential treatment with periodate and (³H)-sodium borohydride, which trititates sialic acid, and by lactoperoxidase-catalyzed (¹²⁵I) iodination of tyrosine residues. The labeled subunits were characterized with respect to their affinity for antisera raised against Triton X-100 extracts of placental brush border membranes. The immunochemically reactive components were analyzed by two-dimensional electrophoresis according to a modification of the O'Farrell technique [20] enabling the assignment of estimated M_r? and pI. Of the 33 ³H-labeled brush border subunits present in Triton X-100-solubilized membrane preparations, 18 subunits reacted with antiplacental brush border antisera insolubilized on CNBr-activated Sepharose or in immunoprecipitates. Fourteen of these tritiated subunits were also labeled with ¹²⁵I, confirming that these are glycoproteins. The plasma membranes of normal human liver and microsomes from kidney were examined for the placental brush border glycoprotein subunits by reaction with insolubilized antiplacental brush border antisera and two-dimensional electrophoresis of the reacting tritium-labeled subunits. Comparison of the two-dimensional electrophoretic maps of the immunochemically reacting glycoproteins from liver, kidney, and placenta resulted in the identification of seven placental subunits in common with liver and kidney on the basis of antigenic cross-reactivity, M_r?, and pI. Four placental glycoproteins were not found in the other tissues and are potentially specific to the placenta. Three of the placental subunits were only seen in placenta and kidney. Three of the subunits ran at the dye front and could not be assigned molecular weights. One of the subunits was poorly labeled by tritiation of sialic acid and was not considered.     

Protease cytochemistry in the murine rodent, guinea-pig and marmoset placenta

Plasma membrane and lysosomal proteases, gamma-glutamyl transferase and extracellular matrix proteases were investigated by qualitative cytochemical means in the mature placenta of mice, rats, guinea-pigs and marmosets. These studies revealed similarities, which concerned primarily the lysosomal proteases in different structures of the placenta and all proteases and gamma-glutamyl transferases in the zone of placental shedding. However, species differences predominated. They were observed especially for amino-peptidase A and M, dipeptidyl peptidase IV and gamma-glutamyl transferase in the plasma membranes and extracellular matrix of the placental barrier and decidual cells of all species and the cells of the basal zone in rats and mice. Plasma membrane and extracellular matrix proteases in other parts of the placenta, e.g. the placenta stem of guinea-pigs and basal plate, amniotic and chorionic plate of marmosets occurred only in these species. Elastase substrates hydrolysing endopeptidase I and kallikrein-, thrombin-, plasmin-, plasminogen- and cathepsin B substrates hydrolysing endopeptidase II were not observed in any of these species. A general comparison of the species revealed similarities for the mouse, rat and guinea-pig placental barrier, but not for that of marmosets. The proteases of this zone in the marmoset placenta are more similar to the human situation, but do not correspond to it completely.     

Uptake of dietary retinoids at the maternal-fetal barrier: in vivo evidence for the role of lipoprotein lipase and alternative pathways

Dietary retinoids (vitamin A and its derivatives) contribute to normal embryonic development. However, the mechanism(s) involved in the transfer of recently ingested vitamin A from mother to embryo is not fully understood. We investigated in vivo whether lipoprotein lipase (LPL) facilitates the placental uptake of dietary retinyl ester incorporated in chylomicrons and their remnants and its transfer to the embryo. We examined the effects of both genetic ablation (MCK-L0 mice) and pharmacological inhibition (P-407) of LPL by maintaining wild type and MCK-L0 mice on diets with different vitamin A content or administering them an oral gavage dose of [(3)H]retinol with or without P-407 treatment. We showed that LPL expressed in placenta facilitates uptake of retinoids by this organ and their transfer to the embryo, mainly through its catalytic activity. In addition, through its "bridging function," LPL can mediate the acquisition of nascent chylomicrons by the placenta, although less efficiently. Quantitative real-time PCR and Western blot analysis showed that placental LPL acts in concert with LDL receptor and LRP1. Finally, by knocking out the retinol-binding protein (RBP) gene in the MCK-L0 background (MCK-L0-RBP(-/-) mice) we demonstrated that the placenta acquires dietary retinoids also via the maternal circulating RBP-retinol complex. RBP expressed in the placenta facilitate the transfer of postprandial retinoids across the placental layers toward the embryo.     

Mek1 and Mek2 functions in the formation of the blood placental barrier

The ERK/MAPK signaling pathway is involved in several cellular functions. Inactivation in mice of genes encoding members of this pathway is often associated with embryonic death resulting from abnormal placental development. The placenta is essential for nutritional and gaseous exchanges between maternal and embryonic circulations, as well as for the removal of metabolic wastes. These exchanges take place without direct contact between the two circulations. In mice, the hematoplacental barrier consists in a triple layer of trophoblast cells and endothelial cells of the embryo. MEK1 and MEK2 are double specificity serine-threonine/tyrosine kinases responsible for the activation of ERK1 and ERK2. Mek1 inactivation results in placental anomalies due to trophoblast cell proliferation and differentiation defects leading to severe delays in the development of placenta and causing the death of the embryo. Although Mek2(-/-) mutant mice survived without any apparent phenotype, double heterozygous Mek1(+/-)Mek2(+/-) mutants die during gestation from placental malformations. Together, these data emphasize the crucial role of the ERK/MAPK cascade in the formation of extraembryonic structures.     

Significance of the placental barrier in antenatal viral infections

The placenta provides a significant physical and physiological barrier to prevent fetal infection during pregnancy. Nevertheless, it is at times breached by pathogens and leads to vertical transmission of infection from mother to fetus. This review will focus specifically on the Zika flavivirus, the HIV retrovirus and the emerging SARS-CoV2 coronavirus, which have affected pregnant women and their offspring in recent epidemics. In particular, we will address how viral infections affect the immune response at the maternal-fetal interface and how the placental barrier is physically breached and discuss the consequences of infection on various aspects of placental function to support fetal growth and development. Improved understanding of how the placenta responds to viral infections will lay the foundation for developing therapeutics to these and emergent viruses, to minimise the harms of infection to the offspring.     

Tissue distribution of hepatocyte growth factor receptor and its exclusive down-regulation in a regenerating organ after injury.

Using 125I-labeled hepatocyte growth factor (HGF) as a ligand, we examined the tissue distribution of the HGF receptor in adult rats. Specific binding of 125I-HGF was detected in the plasma membranes of liver, spleen, kidney, lung, adrenal gland, pituitary, and thyroid. Scatchard analysis of HGF binding in liver, spleen, kidney, lung, and adrenal gland revealed the presence of a single class of high affinity receptor with a dissociation constant (Kd) of 20-30 pM. The maximum number of binding sites (Bmax) was determined to be 400-3,000 sites per ng of plasma membrane protein, the highest number being in the liver. Such a wide distribution of a high affinity HGF receptor indicates that HGF may be a multifunctional growth factor, targeting to a variety of organs, and not restricted to liver. After 70% partial hepatectomy, specific binding of 125I-HGF to membranes of the residual liver rapidly decreased, but there was no change in the kidney, lung, and spleen. On the other hand, after unilateral nephrectomy rapid down-regulation of the HGF receptor was clearly evident in the remaining kidney, but not in other organs including the liver. These findings suggest the presence of control mechanisms governing HGF receptor function only in a regenerating organ after injury.     

Infarct-induced chronic heart failure increases bidirectional protein movement across the alveolocapillary barrier

Chronic heart failure (CHF) is associated with adaptive structural changes at the alveolocapillary barrier that may be associated with altered protein permeability. Bidirectional protein movement across the barrier was studied in anesthetized rats with infarct-induced CHF by following (125)I-labeled albumin ((125)I-albumin) flux into the alveoli and the leakage of surfactant protein (SP)-B from the alveoli into the circulation. Three groups were studied: controls [0% left ventricular (LV) infarction], moderate infarct (25-45% LV infarction), and large infarct (>46% LV infarction). Wet and dry lung weights increased in the large infarct group (both P < 0.001), consistent with increased lung water and solid lung tissue. (125)I-albumin flux increased across the endothelial (P < 0.001) and epithelial (P < 0.01) components of the alveolocapillary barrier in the large infarct group. Plasma SP-B increased 23% with moderate infarcts (P < 0.05) and 97% with large infarcts (P < 0.001), independent of alveolar levels. Lavage fluid immune cells (P < 0.01) and myeloperoxidase activity (P < 0.05) increased in the large infarct group, consistent with inflammation. Bidirectional protein movement across the alveolocapillary barrier is increased in CHF, and alveolar inflammation may contribute to this pathophysiological defect.     

Effects of maturation on RNA transcription and protein expression of four MRP genes in human placenta and in BeWo cells

The placenta is a multifunctional organ that protects the fetus from toxic compounds and the MRPs contribute to this function. The expression of MRP1, MRP2, MRP3, and MRP5 was compared in human placental tissue and in BeWo cells by real-time RT-PCR analysis; protein expression was assessed by Western blot. MRP1 and MRP3 were the most abundantly expressed genes in placenta but only MRP1 was highly expressed in the BeWo cells. Expression of MRP1 increased 4-fold in the third as compared with first trimester placental samples, and increased 20-fold with polarization of BeWo cells. MRP2, MRP3, and MRP5 were weakly expressed both in placenta and BeWo cells. Protein expression followed mRNA quantification for MRP1 and MRP5 but not for MRP2 and MRP3. These data indicated that MRP1 and MRP5 increase with trophoblast maturation, suggesting a particular role for these proteins in the organ functional development.     

Protein clearance from the air spaces and lungs of unanesthetized sheep over 144 h

We studied the rate, the routes, and the mechanisms for protein clearance from the air spaces and lungs of 20 unanesthetized sheep over 144 h. We instilled 100 ml of autologous serum labeled with 125I-albumin into one lung. At the end of 24, 48, 96, or 144 h, the lungs were removed and the residual native protein and 125I-albumin in the air spaces were determined by bronchoalveolar lavage. Also the fraction of the instilled 125I-albumin remaining in the rest of the lung was measured in the lung homogenate. Clearance of the 125I-albumin from the lung into the plasma, lymph, thyroid, urine, and feces was also determined. The removal of both the 125I-albumin and the native protein from the air spaces was slow, following a monoexponential decline. The removal rate of the 125I-albumin from the air spaces was slightly but significantly faster (1.6%/h) than the clearance rate of the native protein (0.9%/h). Clearance of the 125I-albumin from the lung also followed a slow monoexponential decline at a rate of 1.4%/h. At all time periods, 75% of the 125I-albumin remaining in the lung was located in the air spaces, thus indicating that the pulmonary epithelium is the principal barrier to protein clearance from the normal lung. Macrophages appeared to play a minor role in alveolar protein clearance because the quantity of 125I-albumin present in the phagocytic cells in the air spaces was less than 1% of the instilled 125I-albumin at all time periods. However, macrophages may play some role in protein clearance after 48 h because we visualized phagolysosomes in macrophages, and there was an increase in free iodine in lung lavage, urine, thyroid, and feces after 48 h. However, gel electrophoretic studies showed that most of the 125I-albumin was cleared from the lung as an intact molecule, although only 24.7 +/- 4.7% of the 125I-albumin was cleared by the lymphatics.     

Isolation of the hemopexin receptor from human placenta

A hemopexin receptor detected in detergent-solubilized placental membranes was purified from the human placenta, using hemopexin-Sepharose affinity chromatography. The solubilized membranes exhibited binding sites of 2.77 pmol of hemopexin/mg of protein with a dissociation constant (Kd) of 6.6 X 10(-8) M. The purified receptor has a molecular weight of 80,000, determined on sodium dodecyl sulfate-gel electrophoresis. Immunoinhibition experiments using the antibody against the placental receptor revealed inhibition of binding of 125I-hemopexin to human leukemia K562 and HL 60 cells, thereby strongly supporting that the polypeptide isolated from the human placenta was the hemopexin receptor.     

Characterization of Corticotropin-Releasing Hormone Binding Sites in the Human Placenta

Abstract

The human placenta synthesizes and secretes large amounts of corticotropin-releasing hormone (CRH) which has been implicated in the triggering of parturition. The placental CRH was found to act in a paracrine manner to stimulate secretion of ACTH and β-endorphin. In view of this we sought to characterize CRH binding sites in the human placenta.

The specific binding of ¹²⁵I-tyrosyl-ovine CRH (¹²⁵I-oCRH) to placental membranes was dependent on time, temperature, pH, divalent cations and was reversible on addition of excess oCRH. Scatchard analysis revealed a high affinity binding site with a dissociation constant of ?0.7 nmol/L and maximum number of binding sites ?44 fmol/mg protein.

Disuccinimidyl suberate, a chemical cross-linker, was used to covalently attach ¹²⁵I-oCRH to placental membranes. The labelled placental membranes were analyzed by SDS-PAGE and autoradiography. A major radioactively labelled band with a molecular weight of 55,000 Da was identified.

In this study we have identified placental binding sites for CRH with properties similar to CRH receptors described in a number of human and animal tissues and with a molecular weight similar to that of the brain CRH receptor. These binding sites may be involved in the regulation of the placental CRH/ACTH - β-endorphin axis during pregnancy and parturition.     

Quantitative characteristics of components of the placental barrier of women with uncomplicated full-term pregnancies]

Placentas from four healthy women have been studied electron microscopically after urgent spontaneous deliveries. In every placenta 130-200 resorptive villi of the chorion were photographed. The results on morphometric analysis of 1,205 electronograms of the placental barrier are presented. At full-term uncomplicated pregnancies all components of the placental barrier (syncytium, cytotrophoblast, basal membrane of the trophoblast, stromal connective-tissue layer, basal membrane and endothelium of fetal capillaries) have been stated to demonstrate rather stable quantitative characteristics (see Table in the text). The quantitative data obtained can serve for electron microscopic investigation of placenta at different pathologic states of the mother and the fetus, make it possible to differentiate more precisely physiological and pathological changes in placental ultrastructure.     

Strain-specific spleen remodelling in Plasmodium yoelii infections in Balb/c mice facilitates adherence and spleen macrophage-clearance escape

Knowledge of the dynamic features of the processes driven by malaria parasites in the spleen is lacking. To gain insight into the function and structure of the spleen in malaria, we have implemented intravital microscopy and magnetic resonance imaging of the mouse spleen in experimental infections with non-lethal (17X) and lethal (17XL) Plasmodium yoelii strains. Noticeably, there was higher parasite accumulation, reduced motility, loss of directionality, increased residence time and altered magnetic resonance only in the spleens of mice infected with 17X. Moreover, these differences were associated with the formation of a strain-specific induced spleen tissue barrier of fibroblastic origin, with red pulp macrophage-clearance evasion and with adherence of infected red blood cells to this barrier. Our data suggest that in this reticulocyte-prone non-lethal rodent malaria model, passage through the spleen is different from what is known in other Plasmodium species and open new avenues for functional/structural studies of this lymphoid organ in malaria.     

An atypical vasopermeability response to leukotrienes in the iris-ciliary body.

The effects of leukotrienes (LTs) on the blood-aqueous barrier in the guinea pig were investigated. Leukotriene B4 (LTB4) and D4 (LTD4) were administered in doses of 1 to 1000 ng into the anterior chamber. Plasma extravasation was determined as extravascular 125I-albumin levels in the iris-ciliary body using 125I-albumin and 51Cr-erythrocytes as an intravascular marker. LTB4 and LTD4 alone did not cause significant plasma extravasation. Although combinations of 100 ng and more of each LT raised extravascular albumin levels in the ocular anterior uvea. These studies suggest a synergistic interaction of LTB4 and LTD4 with respect to plasma extravasation in the iris-ciliary body. These results further support a possible involvement of LTs in uveal inflammatory diseases.     

Transepidermal water loss: the signal for recovery of barrier structure and function

Previous studies have demonstrated that perturbations in barrier function stimulate epidermal lipid synthesis and that this increase can be prevented by occlusive membranes. These observations suggest that epidermal lipid synthesis might be related to barrier function and raised the question whether transcutaneous water flux might regulate epidermal lipogenesis. In the present study we first abrogated the barrier with acetone, and then compared the rate of repletion of stainable lipids, barrier recovery, and epidermal lipogenesis in animals covered with occlusive membranes or vapor-permeable membranes versus uncovered animals. Acetone treatment of hairless mice removed stainable neutral lipids from the stratum corneum, with repletion evident both biochemically and histochemically within 48 hr in uncovered animals. In contrast, when the animals were covered with an occlusive membrane, the usual return of stratum corneum lipids was aborted. Since application of vapor-permeable membranes allowed normal lipid repletion, occlusion alone is not responsible for the inhibition of lipid repletion. Acetone treatment also perturbed epidermal barrier function, which returned to normal in uncovered animals in parallel with the reappearance of stratum corneum lipid. However, when animals were covered with an occlusive membrane, barrier function did not recover normally. In contrast, occlusion with vapor-permeable membranes allowed barrier function to recover normally. Finally, whereas occlusive membranes prevented the characteristic increase in epidermal lipid synthesis that follows barrier perturbation, epidermal lipid synthesis was increased in animals covered with a vapor-permeable membrane. These results point to transepidermal water flux itself as the signal that regulates epidermal lipid synthesis, which is associated first with the redeposition of stratum corneum lipids and then the normalization of stratum corneum barrier function.