scholarly journals Suppressyn localization and dynamic expression patterns in primary human tissues support a physiologic role in human placentation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jun Sugimoto ◽  
Danny J. Schust ◽  
Tadatsugu Kinjo ◽  
Yoichi Aoki ◽  
Yoshihiro Jinno ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Secretory Pathway ◽  
Ex Vivo ◽  
Expression Patterns ◽  
Extravillous Trophoblast ◽  
Human Trophoblast ◽  
Dynamic Expression ◽  
Ambient Oxygen ◽  
Related Proteins

AbstractWe previously identified suppressyn (SUPYN), a placental protein that negatively regulates the cell fusion essential for trophoblast syncytialization via binding to the trophoblast receptor for syncytin-1, ASCT2, and hypothesized that SUPYN may thereby regulate cell-cell fusion in the placenta. Here, we redefine in vivo SUPYN localization using specific monoclonal antibodies in a rare early placental sample, showing SUPYN localization in villous and extravillous trophoblast subtypes, the decidua and even in placental debris in the maternal vasculature. In human trophoblast cell lines, we show SUPYN alters ASCT2 glycosylation within the secretory pathway and that this binding is associated with inhibition of cell fusion. Using newly-optimized trophoblast isolation protocols that allow tracking of ex vivo cell fusion, we present transcription and translation dynamics of fusion-related proteins over 96 hours in culture and the effects of changes in ambient oxygen levels on these processes. We report converse syncytin-1 and SUPYN transcriptional and translational responses to surrounding oxygen concentrations that suggest both are important in the effects of hypoxia and hyperoxia on placental syncytialization. Our results suggest that SUPYN’s anti-fusogenic properties may be exerted at several sites in the maternal body and its dysregulation may be associated with diseases of abnormal placentation.

PPARγ controls pregnancy outcome through activation of EG-VEGF: new insights into the mechanism of placental development

2015 ◽  
Vol 309 (4) ◽  
pp. E357-E369 ◽  
Author(s):  
Vanessa Garnier ◽  
Wael Traboulsi ◽  
Aude Salomon ◽  
Sophie Brouillet ◽  
Thierry Fournier ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Trophoblast Cell ◽  
Extravillous Trophoblast ◽  
Trophoblast Invasion ◽  
Migration And Invasion ◽  
Placental Development ◽  
Human Trophoblast ◽  
Pparγ Agonist

PPARγ-deficient mice die at E9.5 due to placental abnormalities. The mechanism by which this occurs is unknown. We demonstrated that the new endocrine factor EG-VEGF controls the same processes as those described for PPARγ, suggesting potential regulation of EG-VEGF by PPARγ. EG-VEGF exerts its functions via prokineticin receptor 1 (PROKR1) and 2 (PROKR2). This study sought to investigate whether EG-VEGF mediates part of PPARγ effects on placental development. Three approaches were used: 1) in vitro, using human primary isolated cytotrophoblasts and the extravillous trophoblast cell line (HTR-8/SVneo); 2) ex vivo, using human placental explants ( n = 46 placentas); and 3) in vivo, using gravid wild-type PPARγ+/− and PPARγ−/− mice. Major processes of placental development that are known to be controlled by PPARγ, such as trophoblast proliferation, migration, and invasion, were assessed in the absence or presence of PROKR1 and PROKR2 antagonists. In both human trophoblast cell and placental explants, we demonstrated that rosiglitazone, a PPARγ agonist, 1) increased EG-VEGF secretion, 2) increased EG-VEGF and its receptors mRNA and protein expression, 3) increased placental vascularization via PROKR1 and PROKR2, and 4) inhibited trophoblast migration and invasion via PROKR2. In the PPARγ−/− mouse placentas, EG-VEGF levels were significantly decreased, supporting an in vivo control of EG-VEGF/PROKRs system during pregnancy. The present data reveal EG-VEGF as a new mediator of PPARγ effects during pregnancy and bring new insights into the fine mechanism of trophoblast invasion.

scholarly journals A novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Md Imam Uddin ◽  
Tyler C. Kilburn ◽  
Sara Z. Jamal ◽  
Craig L. Duvall ◽  
John S. Penn
Keyword(s):  
Real Time ◽  
Disease Burden ◽  
Ex Vivo ◽  
Expression Patterns ◽  
Fluorescence Emission ◽  
High Sensitivity ◽  
Living Systems ◽  
Specific Cell ◽  
Potential Marker

AbstractDiabetic retinopathy, retinopathy of prematurity and retinal vein occlusion are potentially blinding conditions largely due to their respective neovascular components. The development of real-time in vivo molecular imaging methods, to assess levels of retinal neovascularization (NV), would greatly benefit patients afflicted with these conditions. mRNA hybridization techniques offer a potential method to image retinal NV. The success of these techniques hinges on the selection of a target mRNA whose tissue levels and spatial expression patterns correlate closely with disease burden. Using a model of oxygen-induced retinopathy (OIR), we previously observed dramatic increases in retinal endoglin that localized to neovascular structures (NV), directly correlating with levels of neovascular pathology. Based on these findings, we have investigated Endoglin mRNA as a potential marker for imaging retinal NV in OIR mice. Also of critical importance, is the application of innovative technologies capable of detecting mRNAs in living systems with high sensitivity and specificity. To detect and visualize endoglin mRNA in OIR mice, we have designed and synthesized a novel imaging probe composed of short-hairpin anti-sense (AS) endoglin RNA coupled to a fluorophore and black hole quencher (AS-Eng shRNA). This assembly allows highly sensitive fluorescence emission upon hybridization of the AS-Eng shRNA to cellular endoglin mRNA. The AS-Eng shRNA is further conjugated to a diacyl-lipid (AS-Eng shRNA–lipid referred to as probe). The lipid moiety binds to serum albumin facilitating enhanced systemic circulation of the probe. OIR mice received intraperitoneal injections of AS-Eng shRNA–lipid. Ex vivo imaging of their retinas revealed specific endoglin mRNA dependent fluorescence superimposed on neovascular structures. Room air mice receiving AS-Eng shRNA–lipid and OIR mice receiving a non-sense control probe showed little fluorescence activity. In addition, we found that cells in neovascular lesions labelled with endoglin mRNA dependent fluorescence, co-labelled with the macrophage/microglia-associated marker IBA1. Others have shown that cells expressing macrophage/microglia markers associate with retinal neovascular structures in proportion to disease burden. Hence we propose that our probe may be used to image and to estimate the levels of retinal neovascular disease in real-time in living systems.

scholarly journals Transcriptional heterogeneity in cancer-associated regulatory T cells is predictive of survival

2018 ◽  
Author(s):  
Nicholas Borcherding ◽  
Kawther K. Ahmed ◽  
Andrew P. Voigt ◽  
Ajaykumar Vishwakarma ◽  
Ryan Kolb ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Immune Cells ◽  
Ex Vivo ◽  
Expression Patterns ◽  
Suppressive Effect ◽  
Renal Clear Cell Carcinoma ◽  
Cell Fates ◽  
Treg Subset

Regulatory T cells (Tregs) are a population of T cells that exert a suppressive effect on a variety of immune cells and non-immune cells. The suppressive effects of Tregs are detrimental to anti-tumor immunity. Recent investigations into cancer-associated Tregs have identified common expression patterns for tumor-infiltration, however the functional heterogeneity in tumor-infiltrating (TI) Treg is largely unknown. We performed single-cell sequencing on immune cells derived from renal clear cell carcinoma (ccRCC) patients, isolating 160 peripheral-blood (PB) Tregs and 574 TI Tregs. We identified distinct transcriptional TI Treg cell fates, with a suppressive subset expressing CD177. We demonstrate CD177+ TI-Tregs have preferential suppressive effects in vivo and ex vivo. Gene signatures derived the CD177+ Treg subset had superior ability to predict survival in ccRCC and seven other cancer types. Further investigation into the development and regulation of TI-Treg heterogeneity will be vital to the application of tumor immunotherapies that possess minimal side effects.

scholarly journals Gene Deletion Mutants Reveal a Role for Semaphorin Receptors of the Plexin-B Family in Mechanisms Underlying Corticogenesis

2009 ◽  
Vol 30 (3) ◽  
pp. 764-780 ◽  
Author(s):  
A. Hirschberg ◽  
S. Deng ◽  
A. Korostylev ◽  
E. Paldy ◽  
M. R. Costa ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Ex Vivo ◽  
Time Windows ◽  
Expression Patterns ◽  
Cortical Development ◽  
Critical Time ◽  
Dependent Manner ◽  
Semaphorin 4D ◽  
Developing Neurons

ABSTRACT Semaphorins and their receptors, plexins, are emerging as key regulators of various aspects of neural and nonneural development. Semaphorin 4D (Sema4D) and B-type plexins demonstrate distinct expression patterns over critical time windows during the development of the murine neocortex. Here, analysis of mice genetically lacking plexin-B1 or plexin-B2 revealed the significance of Sema4D-plexin-B signaling in cortical development. Deficiency of plexin-B2 resulted in abnormal cortical layering and defective migration and differentiation of several subtypes of cortical neurons, including Cajal-Retzius cells, GABAergic interneurons, and principal cells in vivo. In contrast, a lack of plexin-B1 did not impact on cortical development in vivo. In various ex vivo assays on embryonic forebrain, Sema4D enhanced the radial and tangential migration of developing neurons in a plexin-B2-dependent manner. These results suggest that Sema4D-plexin-B2 interactions regulate mechanisms underlying cell specification, differentiation, and migration during corticogenesis.

scholarly journals Dynamic expression pattern and subcellular localization of the Rhox10 homeobox transcription factor during early germ cell development

Reproduction ◽  
2012 ◽  
Vol 143 (5) ◽  
pp. 611-624 ◽  
Author(s):  
Hye-Won Song ◽  
Christina T Dann ◽  
John R McCarrey ◽  
Marvin L Meistrich ◽  
Gail A Cornwall ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Germ Cells ◽  
Expression Patterns ◽  
Homeobox Gene ◽  
Germline Stem Cells ◽  
Dynamic Expression ◽  
Homeobox Transcription Factor ◽  
Dramatic Shift

Homeobox genes encode transcription factors that regulate diverse developmental events. The largest known homeobox gene cluster – the X-linked mouse reproductive homeobox (Rhox) cluster – harbors genes whose expression patterns and functions are largely unknown. Here, we report that a member of this cluster, Rhox10, is expressed in male germ cells. Rhox10 is highly transcribed in spermatogonia in vivo and is upregulated in response to the differentiation-inducing agent retinoic acid in vitro. Using a specific RHOX10 antiserum that we generated, we found that RHOX10 protein is selectively expressed in fetal gonocytes, germline stem cells, spermatogonia, and early spermatocytes. RHOX10 protein undergoes a dramatic shift in subcellular localization as germ cells progress from mitotically arrested gonocytes to mitotic spermatogonia and from mitotic spermatogonia to early meiotic spermatocytes, consistent with RHOX10 performing different functions in these stages.

scholarly journals Inflammatory Bowel Disease–associated GP2 Autoantibodies Inhibit Mucosal Immune Response to Adherent-invasive Bacteria

2020 ◽  
Vol 26 (12) ◽  
pp. 1856-1868
Author(s):  
Stefanie Derer ◽  
Ann-Kathrin Brethack ◽  
Carlotta Pietsch ◽  
Sebastian T Jendrek ◽  
Thomas Nitzsche ◽  
...  
Keyword(s):  
Inflammatory Bowel Disease ◽  
Bowel Disease ◽  
Intestinal Inflammation ◽  
Ex Vivo ◽  
Expression Patterns ◽  
Mucosal Immune Response ◽  
Disease Manifestation ◽  
Inflammatory Bowel

Abstract Adherent-invasive Escherichia coli have been suggested to play a pivotal role within the pathophysiology of inflammatory bowel disease (IBD). Autoantibodies against distinct splicing variants of glycoprotein 2 (GP2), an intestinal receptor of the bacterial adhesin FimH, frequently occur in IBD patients. Hence, we aimed to functionally characterize GP2-directed autoantibodies as a putative part of IBD’s pathophysiology. Ex vivo, GP2-splicing variant 4 (GP2#4) but not variant 2 was expressed on intestinal M or L cells with elevated expression patterns in IBD patients. The GP2#4 expression was induced in vitro by tumor necrosis factor (TNF)-α. The IBD-associated GP2 autoantibodies inhibited FimH binding to GP2#4 and were decreased in anti-TNFα-treated Crohn’s disease patients with ileocolonic disease manifestation. In vivo, mice immunized against GP2 before infection with adherent-invasive bacteria displayed exacerbated intestinal inflammation. In summary, autoimmunity against intestinal expressed GP2#4 results in enhanced attachment of flagellated bacteria to the intestinal epithelium and thereby may drive IBD’s pathophysiology.

scholarly journals Dynamic CCN3 expression in the murine CNS does not confer essential roles in myelination or remyelination

2020 ◽  
Vol 117 (30) ◽  
pp. 18018-18028
Author(s):  
Nira de la Vega Gallardo ◽  
Rosana Penalva ◽  
Marie Dittmer ◽  
Michelle Naughton ◽  
John Falconer ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Ex Vivo ◽  
Matricellular Protein ◽  
Suprachiasmatic Nuclei ◽  
Dynamic Expression ◽  
Ccn3 Expression ◽  
Cns Myelination

CCN3 is a matricellular protein that promotes oligodendrocyte progenitor cell differentiation and myelination in vitro and ex vivo. CCN3 is therefore a candidate of interest in central nervous system (CNS) myelination and remyelination, and we sought to investigate the expression and role of CCN3 during these processes. We found CCN3 to be expressed predominantly by neurons in distinct areas of the CNS, primarily the cerebral cortex, hippocampus, amygdala, suprachiasmatic nuclei, anterior olfactory nuclei, and spinal cord gray matter. CCN3 was transiently up-regulated following demyelination in the brain of cuprizone-fed mice and spinal cord lesions of mice injected with lysolecithin. However, CCN3−/−mice did not exhibit significantly different numbers of oligodendroglia or differentiated oligodendrocytes in the healthy or remyelinating CNS, compared to WT controls. These results suggest that despite robust and dynamic expression in the CNS, CCN3 is not required for efficient myelination or remyelination in the murine CNS in vivo.

Single Dose Administration of rFVIIa and NN1731 in Two Hemophilia A Dogs.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3144-3144 ◽  
Author(s):  
Mirella Ezban ◽  
Lone Frost ◽  
Dorthe Viuff ◽  
Judi Møss ◽  
Mark Kloos ◽  
...  
Keyword(s):  
Hemophilia A ◽  
Ex Vivo ◽  
Compartment Model ◽  
Initial Distribution ◽  
Coagulation Assays ◽  
Two Compartment Model ◽  
Dose Study ◽  
Related Proteins

Abstract Introduction: The objective of this pilot study was to evaluate and compare the pharmacokinetic and pharmacodynamic (PK/PD) profile of rFVIIa and NN1731 in two hemophilia A dogs. In addition, it was the aim to evaluate the use of TEG for monitoring rFVIIa/NN1731 activity after in vivo administration and to compare with ex vivo spiking data from a previous study. NN1731 is a new rFVIIa analoge with enhanced activity (Allen et al. Arterioscler. Thromb. Vasc. Biol.2007;27:683–689). In hemophilia patients as well as hemophilia dogs the clot formation is impaired and reflected in coagulation assays such as thromboelastography (TEG) and APTT. The choice of hemophilia dogs is based upon the knowledge that the pharmacokinetics of human coagulations factors (FVIII, FIX and rFVIIa) as well as the effective dose is similar to that in humans. In normal dogs, it is not possible to evaluate the effect of these procoagulant proteins in coagulation assays as no impaired clotting is observed. Methods: rFVIIa and NN1731 (280 μg/kg IV) were administered to two hemophilia dogs on separate days and plasma samples collected at different time points. FVIIa activity was measured by the FVIIa clot assay and values were used for pharmacokinetic assessment. The same pharmacokinetic models, a non-compartmental method and a two compartment model, respectively, were used as was the case in the First Human Dosing (FHD) trial of NN1731 (NN1731–1639). Analysis of PD markers in dogs included: APTT, PT and whole blood thromboelastography analysis, recently developed for use in hemophilia dogs. Results: Based on the FVIIa activity profile in the two dogs it was observed that the values obtained at the first time point (C5 min), were higher after treatment with NN1731 than after rFVIIa. All activity based assays including TEG demonstrated that NN1731 was cleared faster than rFVIIa., FVIIa activity (FVIIa clot assay), showed a rapid initial distribution and/or elimination of FVIIa activity (t1/2α:0.3 h) followed by a less rapid elimination phase (t½β:3.5 h). Similar profile and values were obtained for NN1731 in the FHD dose study (J. Møss et al, ISTH, 2007) Conclusions: This study indicates that in hemophilia A dogs, NN1731 and rFVIIa have distinct PK profiles and very similar to what is observed in man. All activity assays show the same qualitative profile, the FVIIa clot assay being the most sensitive assay. The TEG data obtained in vivo are in accordance with the values obtained after in vitro spiking. The data support the use of hemophilia dogs for evaluating the pharmacokinetic and pharmacodynamic profiles of FVIIa related proteins.

scholarly journals Lung organoids: advances in generation and 3D-visualization

2021 ◽  
Author(s):  
Brian Cunniff ◽  
Joseph E. Druso ◽  
Jos L. van der Velden
Keyword(s):  
Lung Function ◽  
Ex Vivo ◽  
3D Visualization ◽  
Expression Patterns ◽  
Cell Types ◽  
Experimental Manipulation ◽  
Molecular Response ◽  
Disease States ◽  
And Function

AbstractThe lung is comprised of more than 40 distinct cell types that support a complex 3-dimensional (3D) architecture that is required for efficient lung function. Loss of this proper architecture can accommodate and promote lung disease, highlighting researchers’ growing need to analyze lung structures in detail. Additionally, in vivo cellular and molecular response to chemical and physical signals, along with the recapitulation of gene-expression patterns, can be lost during the transition from complex 3D tissues to 2D cell culture systems. Therefore, technologies that allow for the investigation of lung function under normal and disease states utilizing the entirety of the lung architecture are required to generate a complete understanding of these processes. Airway cell-derived organoids that can recapitulate lung structure and function ex vivo while being amenable to experimental manipulation, have provided a new and exciting model system to investigate lung biology. In this perspective, we discuss emerging technologies for culturing lung-derived organoids, techniques to visualize organoids using high-resolution microscopy and the resulting information extracted from organoids supporting research focused on lung function and diseases.

scholarly journals Spatially-Resolved Live Cell Tagging and Isolation Using Protected Photoactivatable Cell Dyes

2020 ◽  
Author(s):  
Alex S Genshaft ◽  
Carly G. K. Ziegler ◽  
Constantine N. Tzouanas ◽  
Benjamin E. Mead ◽  
Alex M. Jaeger ◽  
...  
Keyword(s):  
Immune Cell ◽  
Lung Tumor ◽  
Uv Light ◽  
Ex Vivo ◽  
Expression Patterns ◽  
Temporal Stability ◽  
Tumor Model ◽  
Dynamic Interactions

ABSTRACTWhether cultured in vitro or part of a complex tissue in vivo, a cell’s phenotype and function are significantly influenced by dynamic interactions with its microenvironment. To explicitly examine how a cell’s spatiotemporal activity impacts its behavior, we developed and validated a strategy termed SPACECAT—Spatially PhotoActivatable Color Encoded Cell Address Tags—to annotate, track, and isolate specific cells in a non-destructive, viability-preserving manner. In SPACECAT, a biological sample is immersed in a photocaged fluorescent molecule, and cells within a location of interest are labeled for further study by uncaging that molecule with user-patterned near-UV light. SPACECAT offers high spatial precision and temporal stability across diverse cell and tissue types, and is compatible with common downstream assays, including flow cytometry and single-cell RNA-Seq. Illustratively, we leveraged this approach in patient-derived intestinal organoids, a spatially complex system less amenable to genetic manipulations, to select for crypt-like regions enriched in stem-like and actively mitotic cells. Moreover, we demonstrate its applicability and utility on ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model, uncovering spatially-biased gene expression patterns among immune cell subsets and identifying rare myeloid phenotypes enriched around tumor/healthy border regions. In sum, our method provides a minimally invasive and broadly applicable approach to link cellular spatiotemporal features and/or behavioral phenotypes with diverse downstream assays, enabling fundamental insights into the connections between tissue microenvironments and biological (dys)function.

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