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Susan J Fisher

Publications and source records attributed to Susan J Fisher.

At least 19 recordsLinked to original sources
PubMed2006

Gene expression profiling of the human maternal-fetal interface reveals dramatic changes between midgestation and term.

Human placentation entails the remarkable integration of fetal and maternal cells into a single functional unit. In the basal plate region (the maternal-fetal interface) of the placenta, fetal cytotrophoblasts from the placenta invade the uterus and remodel the resident vasculature and avoid maternal immune rejection. Knowing the molecular bases for these unique cell-cell interactions is important for understanding how this specialized region functions during normal pregnancy with implications for tumor biology and transplantation immunology. Therefore, we undertook a global analysis of the gene expression profiles at the maternal-fetal interface. Basal plate biopsy specimens were obtained from 36 placentas (14-40 wk) at the conclusion of normal pregnancies. RNA was isolated, processed, and hybridized to HG-U133A&B Affymetrix GeneChips. Surprisingly, there was little change in gene expression during the 14- to 24-wk interval. In contrast, 418 genes were differentially expressed at term (37-40 wk) as compared with midgestation (14-24 wk). Subsequent analyses using quantitative PCR and immunolocalization approaches validated a portion of these results. Many of the differentially expressed genes are known in other contexts to be involved in differentiation, motility, transcription, immunity, angiogenesis, extracellular matrix dissolution, or lipid metabolism. One sixth were nonannotated or encoded hypothetical proteins. Modeling based on structural homology revealed potential functions for 31 of these proteins. These data provide a reference set for understanding the molecular components of the dialogue taking place between maternal and fetal cells in the basal plate as well as for future comparisons of alterations in this region that occur in obstetric complications.

Female↗
PubMed2006

Cytotrophoblast induction of arterial apoptosis and lymphangiogenesis in an in vivo model of human placentation.

We studied the vascular effects of invasive human cytotrophoblasts in vivo by transplanting placental villi to the fifth mammary fat pads or beneath the kidney capsules of Scid mice. Over 3 weeks, robust cytotrophoblast invasion was observed in both locations. The architecture of the mammary fat pad allowed for detailed analysis of the cells' interactions with resident murine blood vessels, which revealed specific induction of apoptosis in the endothelial cells and smooth muscle walls of the arterioles. This finding, and confirmation of the results in an in vitro coculture model, suggests that a parallel process is important for enabling cytotrophoblast endovascular invasion during human pregnancy. Cytotrophoblast invasion of the kidney parenchyma was accompanied by a robust lymphangiogenic response, while in vitro, the cells stimulated lymphatic endothelial cell migration via the actions of VEGF family members, FGF, and TNF-alpha. Immunolocalization analyses revealed that human pregnancy is associated with lymphangiogenesis in the decidua since lymphatic vessels were not a prominent feature of the nonpregnant endometrium. Thus, the placenta triggers the development of a decidual lymphatic circulation, which we theorize plays an important role in maintaining fluid balance during pregnancy, with possible implications for maternal-fetal immune cell trafficking.

Animals↗
PubMed2006

MUC1 is a scaffold for selectin ligands in the human uterus.

MUC1 is a large, transmembrane mucin glycoprotein abundantly expressed at the apical surface of uterine epithelia in all species examined to date. Loss of MUC1 at the time of embryo implantation occurs in many species; however, this does not appear to be the case in humans. Recent studies indicate that human blastocysts express L-selectin at their external surfaces raising the possibility that selectin ligands expressed at the apical surface of the uterine epithelium support early stages of blastocyst attachment. In the current study, we have used a panel of antibodies specific for selectin ligands to determine if MUC1 functions as a scaffold for these carbohydrate motifs in fertile women. The results demonstrate that MUC1 carries selectin ligands throughout the secretory phase of the menstrual cycle, including the mid-secretory (receptive) phase. Consequently, MUC1 represents a potential ligand for selectins expressed by human blastocysts.

Antibodies↗
PubMed2006

Nicotine downregulates the l-selectin system that mediates cytotrophoblast emigration from cell columns and attachment to the uterine wall.

Here we show that maternal smoking downregulated, in a dose-dependent manner, cytotrophoblast expression of l-selectin and its TRA-1-81-reactive carbohydrate ligands. Cell islands -- cell columns that fail to make uterine attachments, often more numerous in the placentas of smokers -- exhibited an even greater downregulation of the l-selectin adhesion system. These effects were attributable to nicotine, since exposure of explanted villi to this drug in vitro reproduced the effects observed in situ. Videomicroscopy showed that the downstream consequences included inhibition of all stages of cytotrophoblast outgrowth from columns, including rolling adhesion within columns and generation of invasive cells at the distal ends. These results suggest that nicotine, acting through the l-selectin adhesion system, impairs the development of cell columns that connect the fetal portion of the placenta to the uterus, one possible reason why women who smoke have a much harder time achieving and sustaining pregnancy than their nonsmoking counterparts.

Antibodies, Monoclonal↗
PubMed2006

A role for the L-selectin adhesion system in mediating cytotrophoblast emigration from the placenta.

Cytotrophoblast (CTB) aggregates that bridge the gap between the placenta and the uterus are suspended as cell columns in the intervillous space, where they experience significant amounts of shear stress generated by maternal blood flow. The proper formation of these structures is crucial to pregnancy outcome as they play a vital role in anchoring the embryo/fetus to the decidua. At the same time, they provide a route by which CTBs enter the uterine wall. The mechanism by which the integrity of the columns is maintained while allowing cell movement is unknown. Here, we present evidence that the interactions of L-selectin with its carbohydrate ligands, a specialized adhesion system that is activated by shear stress, play an important role. CTBs in cell columns, particularly near the distal ends, stained brightly for L-selectin and with the TRA-1-81 antibody, which recognizes carbohydrate epitopes that support binding of L-selectin chimeras in vitro. Function-perturbing antibodies that inhibited either receptor or ligand activity also inhibited formation of cell columns in vitro. Together, these results suggest an autocrine role for the CTB L-selectin adhesion system in forming and maintaining cell columns during the early stages of placental development, when the architecture of the basal plate region is established. This type of adhesion may also facilitate CTB exit from cell columns, a prerequisite for uterine invasion.

Antibodies, Monoclonal↗
PubMed2006

Binding of the streptococcal surface glycoproteins GspB and Hsa to human salivary proteins.

GspB and Hsa are homologous surface glycoproteins of Streptococcus gordonii that bind sialic acid moieties on platelet membrane glycoprotein Ibalpha. Since this species is an important member of the oral flora, we examined the direct binding of these adhesins to human salivary proteins. Both GspB and Hsa bound low-molecular-weight salivary mucin MG2 and salivary agglutinin. Hsa also bound several other salivary proteins, including secretory immunoglobulin A. Screening of six oral streptococcal isolates revealed that at least two of the strains expressed GspB homologues. These results indicate that GspB-like adhesins may be important for oral bacterial colonization.

Adhesins, Bacterial↗
PubMed2005

EPHB4 regulates chemokine-evoked trophoblast responses: a mechanism for incorporating the human placenta into the maternal circulation.

In humans, fetal cytotrophoblasts leave the placenta and enter the uterine wall, where they preferentially remodel arterioles. The fundamental mechanisms that govern these processes are largely unknown. Previously, we have shown that invasive cytotrophoblasts express several chemokines, as well as the receptors with which they interact. Here, we report that these ligand-receptor interactions stimulate cytotrophoblast migration to approximately the same level as a growth factor cocktail that includes serum. Additionally, cytotrophoblast commitment to uterine invasion was accompanied by rapid downregulation of EPHB4, a transmembrane receptor associated with venous identity, and upregulation of ephrin B1. Within the uterine wall, the cells also upregulated expression of ephrin B2, an EPH transmembrane ligand that is associated with arterial identity. In vitro cytotrophoblasts avoided EPHB4-coated substrates; upon co-culture with 3T3 cells expressing this molecule, their migration was significantly inhibited. As to the mechanisms involved, cytotrophoblast interactions with EPHB4 downregulated chemokine-induced but not growth factor-stimulated migration. We propose that EPHB4/ephrin B1 interactions generate repulsive signals that direct cytotrophoblast invasion toward the uterus, where chemokines stimulate cytotrophoblast migration through the decidua. When cytotrophoblasts encounter EPHB4 expressed by venous endothelium, ephrin B-generated repulsive signals and a reduction in chemokine-mediated responses limit their interaction with veins. When they encounter ephrin B2 ligands expressed in uterine arterioles, migration is permitted. The net effect is preferential cytotrophoblast remodeling of arterioles, a hallmark of human placentation.

3T3 Cells↗
PubMed2005

Assessing the effects of diurnal variation on the composition of human parotid saliva: quantitative analysis of native peptides using iTRAQ reagents.

Changes in salivary composition correlate with disease susceptibility, disease state, or both. However, use of saliva for diagnostic purposes is complicated by the gland-specific effects of circadian rhythm or diurnal variation. We recently characterized a suite of peptides in the < or =10-kDa fraction of human parotid saliva that included many novel species. In this study, we used novel iTRAQ labeling chemistry to investigate possible diurnal effects on peptide generation. We collected samples produced by gustatory stimulation as the ductal secretions at four time points under conditions that minimized proteolysis, pooled them according to collection time, and isolated the LMW fractions. Samples collected at each collection time were derivatized with a different isobaric iTRAQ reagent. The labeled samples were combined, separated by reversed-phase HPLC, co-spotted with matrix on MALDI targets, and analyzed by MALDI TOF/TOF mass spectrometry. With this approach, we achieved relative quantification of the parotid peptides at four time points. In several cases, abundance during the day changed dramatically. iTRAQ tagging improved the efficiency of MS/MS fragmentation, which in turn allowed the identification of several novel peptides. Our results demonstrated both the utility of this method and the importance of diurnal effects on the composition of the human parotid saliva peptidome.

Amino Acid Sequence↗
PubMed2005

Hypoxia-inducible factor-dependent histone deacetylase activity determines stem cell fate in the placenta.

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of HIFalpha and the arylhydrocarbon receptor nuclear translocator (ARNT/HIF1beta). Previously, we have reported that ARNT function is required for murine placental development. Here, we used cultured trophoblast stem (TS) cells to investigate the molecular basis of this requirement. In vitro, wild-type TS cell differentiation is largely restricted to spongiotrophoblasts and giant cells. Interestingly, Arnt-null TS cells differentiated into chorionic trophoblasts and syncytiotrophoblasts, as demonstrated by their expression of Tfeb, glial cells missing 1 (Gcm1) and the HIV receptor CXCR4. During this process, a region of the differentiating Arnt-null TS cells underwent granzyme B-mediated apoptosis, suggesting a role for this pathway in murine syncytiotrophoblast turnover. Surprisingly, HIF1alpha and HIF2alpha were induced during TS cell differentiation in 20% O2; additionally, pVHL levels were modulated during the same time period. These results suggest that oxygen-independent HIF functions are crucial to this differentiation process. As histone deacetylase (HDAC) activity has been linked to HIF-dependent gene expression, we investigated whether ARNT deficiency affects this epigenetic regulator. Interestingly, Arnt-null TS cells had reduced HDAC activity, increased global histone acetylation, and altered class II HDAC subcellular localization. In wild-type TS cells, inhibition of HDAC activity recapitulated the Arnt-null phenotype, suggesting that crosstalk between the HIFs and the HDACs is required for normal trophoblast differentiation. Thus, the HIFs play important roles in modulating the developmental plasticity of stem cells by integrating physiological, transcriptional and epigenetic inputs.

Animals↗
PubMed2005

Human cytotrophoblasts acquire aneuploidies as they differentiate to an invasive phenotype.

Through an unusual differentiation process, human trophoblast progenitors (cytotrophoblasts) give rise to tumor-like cells that invade the uterus. By an unknown mechanism, invasive cytotrophoblasts exhibit permanent cell cycle withdrawal. Here, we report molecular cytogenetic data showing that approximately 20 to 60% of these interphase cells had acquired aneusomies involving chromosomes X, Y, or 16. The incidence positively correlated with gestational age and differentiation to an invasive phenotype. Scoring 12 chromosomes in flow-sorted cytotrophoblasts showed that more than 95% of the cells were hyperdiploid. Thus, aneuploidy appears to be an important component of normal placentation, perhaps limiting the proliferative and invasive potential of cytotrophoblasts within the uterus.

Aneuploidy↗
PubMed2005

Toward defining the human parotid gland salivary proteome and peptidome: identification and characterization using 2D SDS-PAGE, ultrafiltration, HPLC, and mass spectrometry.

Saliva plays many biological roles, from lubrication and digestion to regulating bacterial and leukocyte adhesion. To understand the functions of individual components and families of molecules, it is important to identify as many salivary proteins as possible. Toward this goal, we used a proteomic approach as the first step in a global analysis of this important body fluid. We collected parotid saliva as the ductal secretion from three human donors and separated the protein components by two-dimensional SDS-polyacrylamide gel electrophoresis (2D SDS-PAGE). Proteins in gel spots were identified by peptide mass fingerprinting, and the results were confirmed by tandem mass spectrometry of selected peptides. Complementing this approach we used ultrafiltration to prepare a low-molecular-weight fraction of parotid saliva, which was analyzed directly or after reversed phase high-performance liquid chromatography separation by using mass spectrometric approaches. MS analyses of 2D SDS-PAGE spots revealed known components of saliva, including cystatins, histatins, lysozyme, and isoforms and/or fragments of alpha-amylase, albumin, and proline-rich proteins. We also discovered novel proteins, such as several isoforms of Zn-alpha-2-glycoprotein and secretory actin-binding protein. MS analyses of the ultrafiltrate showed that the low-molecular-weight fraction of parotid saliva was peptide-rich, with novel fragments of proline-rich proteins and histatins in abundance. Experiments using Candida albicans as the test organism showed that at least one of the novel peptides had antifungal activity. Our results show that saliva is a rich source of proteins and peptides that are potential diagnostic and therapeutic targets.

Adult↗
PubMed2005

Highly glycosylated human salivary molecules present oligosaccharides that mediate adhesion of leukocytes and Helicobacter pylori.

Glycoproteins display carbohydrate facets that serve as adhesion receptors for cells including leukocytes and bacterial cells. Our aim was to understand the role of the specialized carbohydrate motifs carried by highly glycosylated human salivary proteins in regulating the oral ecology. To date, our structural studies suggest that these molecules display a wide array of oligosaccharide structures, including many species with highly charged and/or fucosylated termini. Here, we used an immunoblot approach to gain additional information about the nature of these oligosaccharides. The results showed that MG1 and the salivary agglutinin express the MECA-79 epitope, an unusual sulfated carbohydrate structure that belongs to an important class of high-affinity (endothelial) L-selectin ligands. Unexpectedly, we discovered that in many women the expression of this epitope is hormonally regulated. Additional experiments revealed that MG1, MG2, and the salivary agglutinin also present Lewis blood group antigens, the exact repertoire varying on an individual basis. In parallel, we explored the functions of these carbohydrate motifs. Using an assay that detects L-selectin ligands, we found that the subset of MECA-79-reactive oligosaccharides displayed on salivary molecules specifically bind an L-selectin/Fc chimera. In contrast, the Lewis blood group structures are receptors for many strains of Helicobacter pylori, an organism that is implicated in the development of gastric ulcers and cancer. Together, these results suggest that MG1, MG2, and the salivary agglutinin play important roles in governing leukocyte and bacterial adhesion. Our findings suggest novel strategies, based on the relevant carbohydrate structures, for promoting or inhibiting these processes.

Adult↗
PubMed2005

Serum-free derivation of human embryonic stem cell lines on human placental fibroblast feeders.

OBJECTIVE: To derive new human embryonic stem cell (hESC) lines on pathogen-free human placental fibroblast feeders under serum-free conditions. Because the embryo develops in close contact with extraembryonic membranes, we hypothesized that placental mesenchyme might replicate the stem cell niche in situ. DESIGN: We isolated and characterized human placental fibroblast lines from individual donors and tested their ability to support growth of federally registered hESC lines. Moreover, we performed extensive pathogen testing to ensure their suitability as feeders for the derivation of therapy-grade hESCs. RESULT(S): Human placental fibroblasts were comparable or superior to mouse embryo fibroblasts as hESC feeders. We used these qualified placental fibroblasts to derive two new hESC lines in knockout Dulbecco's modified Eagle's medium with serum-free 20% knockout serum replacement. The cells, which had a normal karyotype, were grown for more than 25 passages, expressed markers of stemness including Oct-3/4, Tra 1-60, Tra 1-80, and SSEA-4, exhibited high telomerase activity, and differentiated in vitro and in vivo into cells derived from all three germ layers, confirming their pluripotency. Additionally, newly derived hESCs were adapted to growth on a human placental laminin substrate in a defined medium. CONCLUSION(S): To our knowledge, this is the first report of hESC derivation in the absence of serum on qualified pathogen-free human feeders.

Cell Differentiation↗
PubMed2004

Trisomy 21 is associated with variable defects in cytotrophoblast differentiation along the invasive pathway.

Aneuploid cells in the placenta are associated with poor pregnancy outcomes, but the mechanisms are unclear. Here, we examined the cytotrophoblast (CTB) differentiation pathway that leads to uterine invasion in pregnancies complicated by trisomy 21 (T21) as compared with their normal counterparts. Surprisingly, we observed a wide spectrum of T21 effects. Morphologically, some samples appeared near normal, while others had extensive fibrinoid deposition and apoptosis of CTBs at the maternal-fetal interface (confirmed by TUNEL labeling). At a molecular level, the cells' expression of stage-specific molecules was variably misregulated. At one end of the spectrum, samples with less apoptosis had relatively normal staining patterns. At the other end, samples with extensive apoptosis showed significantly decreased staining for these antigens. Additional studies confirmed that the effects we observed had functional consequences, because the cells exhibited marked phenotypic alterations in vitro, including a large increase in MMP-9 production, which distinguishes the effects of T21 on CTBs from those of preeclampsia. The morphologic, phenotypic, and functional differences among CTBs from pregnancies complicated by T21 illustrate the importance of the interplay between fetal/placental genotype and maternal influences on pregnancy outcome. Furthermore, our data may explain why a significant number of these pregnancies end in spontaneous abortion while others survive to term.

Abortion, Spontaneous↗
PubMed2004

Functional and placental expression analysis of the human NRF3 transcription factor.

Members of the Maf protooncogene and cap'n' collar families of basic-leucine zipper transcription factors play important roles in development, differentiation, oncogenesis, and stress signaling. In this study, we performed an in vivo protein-protein interaction screen to search for novel partners of the small Maf proteins. Using full-length human MAFG protein as bait, we identified the human basic-leucine zipper protein NRF3 [NF-E2 (nuclear factor erythroid 2)-related factor 3] as an interaction partner. Transfection studies confirmed that NRF3 is able to dimerize with MAFG. The resulting NRF3/MAFG heterodimer recognizes nuclear factor-erythroid 2/Maf recognition element-type DNA-binding motifs. Functional analysis revealed the presence of a strong transcriptional activation domain in the center region of the NRF3 protein. We found that NRF3 transcripts are present in placental chorionic villi from at least week 12 of gestation on through term. In particular, NRF3 is highly expressed in primary placental cytotrophoblasts, but not in placental fibroblasts. The human choriocarcinoma cell lines BeWo and JAR, derived from trophoblastic tumors of the placenta, also strongly express NRF3 transcripts. We generated a NRF3-specific antiserum and identified NRF3 protein in placental choriocarcinoma cells. Furthermore, we showed that NRF3 transcript and protein levels are induced by TNF-alpha in JAR cells. Our functional studies suggest that human NRF3 is a potent transcriptional activator. Finally, our expression and induction analyses hint at a possible role of Nrf3 in placental gene expression and development.

Amino Acid Sequence↗
PubMed2004

The placental problem: linking abnormal cytotrophoblast differentiation to the maternal symptoms of preeclampsia.

The placenta is a remarkable organ. In normal pregnancy its specialized cells (termed cytotrophoblasts) differentiate into various specialized subpopulations that play pivotal roles in governing fetal growth and development. One cytotrophoblast subset acquires tumor-like properties that allow the cells to invade the decidua and myometrium, a process that attaches the placenta to the uterus. The same subset also adopts a vascular phenotype that allows these fetal cells to breach and subsequently line uterine blood vessels, a process that channels maternal blood to the rest of the placenta. In the pregnancy complication preeclampsia, which is characterized by the sudden onset of maternal hypertension, proteinuria and edema, cytotrophoblast invasion is shallow and vascular transformation incomplete. These findings, together with very recent evidence from animal models, suggest that preeclampsia is associated with abnormal placental production of vasculogenic/angiogenic substances that reach the maternal circulation with the potential to produce at least a subset of the clinical signs of this syndrome. The current challenge is to build on this knowledge to design clinically useful tests for predicting, diagnosing and treating this dangerous disorder.

Angiogenesis Inducing Agents↗
PubMed2004

Human pregnancy: the role of chemokine networks at the fetal-maternal interface.

Chemokines are multifunctional molecules initially described as having a role in leukocyte trafficking and later found to participate in developmental processes such as differentiation and directed migration. Similar events occur in pregnancy during development of the fetal-maternal interface, where there is extensive leukocyte trafficking and tissue morphogenesis, and this is accompanied by abundant chemokine expression. The relationship between chemokines, leukocytes and placental development is beginning to be delineated. During pregnancy a specialised population of maternal leukocytes infiltrates the implantation site. These leukocytes are thought to sustain the delicate balance between protecting the developing embryo/fetus and tolerating its hemiallogeneic tissues. A network of chemokine expression by both fetal and maternal components in the pregnant uterus functions in establishing this leukocyte population. Intriguingly, experiments investigating immune cell recruitment revealed the additional possibility that chemokines influence aspects of placental development. Specifically, cytotrophoblasts, the effector cells of the placenta, express chemokine receptors that can bind ligands found at key locations, implicating chemokines as regulators of cytotrophoblast differentiation and migration. Thus, as in other systems, at the fetal-maternal interface chemokines might regulate multiple functions.

Chemokines↗