Characterization of the adverse effects of nicotine on placental development: in vivo and in vitro studies

In utero exposure to nicotine is associated with increased risk of numerous adverse fetal and neonatal outcomes, which suggests that it acts directly to affect placental development and the establishment of the fetomaternal circulation (FC). This study used both in vivo [Wistar rats treated with 1 mg/kg nicotine from 2 wk prior to mating until gestational day (GD) 15] and in vitro (RCHO-1 cell line; treated with 10−9 to 10−3M nicotine) models to examine the effects of nicotine on these pathways. At GD 15, control and treated placentas were examined for the impact of nicotine on 1) trophoblast invasion, proliferation, and degree of hypoxia, 2) labyrinth vascularization, 3) expression of key genes of placental development, and 4) expression of placental angiogenic factors. The RCHO-1 cell line was used to determine the direct effects of nicotine on trophoblast differentiation. Our in vivo experiments show that nicotine inhibits trophoblast interstitial invasion, increases placental hypoxia, downregulates labyrinth vascularization as well as key transcription factors Hand1 and GCM1, and decreases local and circulating EG-VEGF, a key placental angiogenic factor. The in vitro experiments confirmed the inhibitory effects of nicotine on the trophoblast migration, invasion, and differentiation processes and demonstrated that those effects are most likely due to a dysregulation in the expression of nicotine receptors and a decrease in MMP9 activity. Taken together, these data suggest that adverse effects of maternal smoking on pregnancy outcome are due in part to direct and endocrine effects of nicotine on the main processes of placental development and establishment of FC.

Download Full-text

Silencing VEGF enhances the radiosensitivity of the radioresistant human nasopharyngeal carcinoma cell line CNE-2R by inhibiting autophagy through the activation of mTOR pathway

10.21203/rs.3.rs-26678/v1 ◽

2020 ◽

Author(s):

Li Chen ◽

Guoxiang Lin ◽

Kaihua Chen ◽

Fangzhu Wan ◽

Yongchu Sun ◽

...

Keyword(s):

Dna Damage ◽

Nasopharyngeal Carcinoma ◽

Cell Line ◽

Western Blotting ◽

Xenograft Model ◽

Mtor Pathway ◽

Angiogenic Factor ◽

Cell Counting

Abstract Background: Vascular endothelial growth factor (VEGF) is an important pro-angiogenic factor. VEGF was reported to promote the occurrence of autophagy, which enhanced to the radioresistance of tumors. The purpose of our study was to investigate the influence of VEGF silencing on the radiosensitivity of nasopharyngeal carcinoma radioresistant cell line CNE-2R and the underlying mechanisms.Methods: The radiosensitivity of CNE-2R cells after silencing VEGF was detected by cell counting kit 8 (CCK-8) and clonogenic assay, cell cycle and apoptosis was subjected to flow cytometry. DNA damage and autophagy were observed by immunofluorescence and western blotting. The interaction between VEGF and mTOR was confirmed by western blotting and co-immunoprecipitation analysis. In vivo, the effect of VEGF on radiosensitivity of NPC cells was investigated through xenograft model, furthermore, immunohistochemistry and TUNEL assay were used to further verify the relationship between autophagy and radiosensitivity in NPC after VEGF depletion.Results: Downregulation of VEGF significantly inhibited cell proliferation and induced apoptosis of CNE-2R cells after radiotherapy in vitro and in vivo. In addition, VEGF knockdown not only decreased autophagy level, but also delayed the DNA damage repair in CNE-2R cells after irradiation. Mechanistically, silencing VEGF suppressed autophagy through the activation of mTOR pathway.Conclusion: VEGF depletion increased radiosensitivity of NPC radioresistant cell CNE-2R by suppressing autophagy via the activation of mTOR pathway.

Download Full-text

A switch toward angiostatic gene expression impairs the angiogenic properties of endothelial progenitor cells in low birth weight preterm infants

Blood ◽

10.1182/blood-2010-12-325142 ◽

2011 ◽

Vol 118 (6) ◽

pp. 1699-1709 ◽

Cited By ~ 57

Author(s):

Isabelle Ligi ◽

Stéphanie Simoncini ◽

Edwige Tellier ◽

Paula Frizera Vassallo ◽

Florence Sabatier ◽

...

Keyword(s):

Birth Weight ◽

Low Birth Weight ◽

Profile Analysis ◽

Preterm Neonates ◽

Akt Phosphorylation ◽

Protein Levels ◽

Increased Risk ◽

The Impact

Abstract Low birth weight (LBW) is associated with increased risk of cardiovascular diseases at adulthood. Nevertheless, the impact of LBW on the endothelium is not clearly established. We investigate whether LBW alters the angiogenic properties of cord blood endothelial colony forming cells (LBW-ECFCs) in 25 preterm neonates compared with 25 term neonates (CT-ECFCs). We observed that LBW decreased the number of colonies formed by ECFCs and delayed the time of appearance of their clonal progeny. LBW dramatically reduced LBW-ECFC capacity to form sprouts and tubes, to migrate and to proliferate in vitro. The angiogenic defect of LBW-ECFCs was confirmed in vivo by their inability to form robust capillary networks in Matrigel plugs injected in nu/nu mice. Gene profile analysis of LBW-ECFCs demonstrated an increased expression of antiangiogenic genes. Among them, thrombospondin 1 (THBS1) was highly expressed at RNA and protein levels in LBW-ECFCs. Silencing THBS1 restored the angiogenic properties of LBW-ECFCs by increasing AKT phosphorylation. The imbalance toward an angiostatic state provide a mechanistic link between LBW and the impaired angiogenic properties of ECFCs and allows the identification of THBS1 as a novel player in LBW-ECFC defect, opening new perspectives for novel deprogramming agents.

Download Full-text

Poorly controlled diabetes mellitus alters placental structure, efficiency, and plasticity

BMJ Open Diabetes Research & Care ◽

10.1136/bmjdrc-2020-001243 ◽

2020 ◽

Vol 8 (1) ◽

pp. e001243

Author(s):

Jackson Nteeba ◽

Kaela M Varberg ◽

Regan L Scott ◽

Mikaela E Simon ◽

Khursheed Iqbal ◽

...

Keyword(s):

Diabetes Mellitus ◽

Fetal Development ◽

Trophoblast Invasion ◽

Ambient Conditions ◽

Placental Development ◽

Junctional Zone ◽

Pregnant Rat ◽

Elevated Glucose ◽

The Impact

IntroductionThe hemochorial placenta provides a critical barrier at the maternal–fetal interface to modulate maternal immune tolerance and enable gas and nutrient exchange between mother and conceptus. Pregnancy outcomes are adversely affected by diabetes mellitus; however, the effects of poorly controlled diabetes on placental formation, and subsequently fetal development, are not fully understood.Research design and methodsStreptozotocin was used to induce hyperglycemia in pregnant rats for the purpose of investigating the impact of poorly controlled diabetes on placental formation and fetal development. The experimental paradigm of hypoxia exposure in the pregnant rat was also used to assess properties of placental plasticity. Euglycemic and hyperglycemic rats were exposed to ambient conditions (~21% oxygen) or hypoxia (10.5% oxygen) beginning on gestation day (gd) 6.5 and sacrificed on gd 13.5. To determine whether the interaction of hyperglycemia and hypoxia was directly altering trophoblast lineage development, rat trophoblast stem (TS) cells were cultured in high glucose (25 mM) and/or exposed to low oxygen (0.5% to 1.5%).ResultsDiabetes caused placentomegaly and placental malformation, decreasing placental efficiency and fetal size. Elevated glucose disrupted rat TS cell differentiation in vitro. Evidence of altered trophoblast differentiation was also observed in vivo, as hyperglycemia affected the junctional zone transcriptome and interfered with intrauterine trophoblast invasion and uterine spiral artery remodeling. When exposed to hypoxia, hyperglycemic rats showed decreased proliferation and ectoplacental cone development on gd 9.5 and complete pregnancy loss by gd 13.5. Furthermore, elevated glucose concentrations inhibited TS cell responses to hypoxia in vitro.ConclusionsOverall, these results indicate that alterations in placental development, efficiency, and plasticity could contribute to the suboptimal fetal outcomes in offspring from pregnancies complicated by poorly controlled diabetes.

Download Full-text

Lipopolysaccharide administration alters extracellular vesicles in human lung-cancer cells and mice

10.1101/2020.04.17.046367 ◽

2020 ◽

Author(s):

Leandra B. Jones ◽

Sanjay Kumar ◽

Courtnee’ R. Bell ◽

Brennetta J. Crenshaw ◽

Mamie T. Coats ◽

...

Keyword(s):

Membrane Protein ◽

Bacterial Infection ◽

Cell Line ◽

Extracellular Vesicles ◽

A549 Cells ◽

Human Lung Cancer ◽

Diagnostic Tools ◽

The Impact

AbstractExtracellular vesicles (EVs) play a fundamental role in cell and infection biology and have the potential to act as biomarkers for novel diagnostic tools. In this study, we explored the in vitro impact of bacterial lipopolysaccharide administration on a cell line that represents a target for bacterial infection in the host. Administration of lipopolysaccharide at varying concentrations to this A549 cell line caused only modest changes in cell death, but EV numbers were significantly changed. After treatment with the highest concentration of lipopolysaccharide, EVs derived from A549 cells packaged significantly less interleukin-6 and lysosomal-associated membrane protein 1. We also examined the impact of lipopolysaccharide administration on exosome biogenesis and cargo composition in BALB/c mice. Serum-isolated EVs from lipopolysaccharide-treated mice showed significantly increased lysosomal-associated membrane protein 1 and toll-like receptor 4 levels compared with EVs from control mice. In summary, this study demonstrated that EV numbers and cargo were altered using these in vitro and in vivo models of bacterial infection.

Download Full-text

The Impact of the Extracellular Matrix Environment on Sost Expression by the MLO-Y4 Osteocyte Cell Line

Bioengineering ◽

10.3390/bioengineering9010035 ◽

2022 ◽

Vol 9 (1) ◽

pp. 35

Author(s):

Robert T. Brady ◽

Fergal J. O’Brien ◽

David A. Hoey

Keyword(s):

Extracellular Matrix ◽

Cell Line ◽

Three Dimensional ◽

3D Culture ◽

Specific Gene ◽

Collagen Scaffolds ◽

Sost Gene ◽

The Impact

Bone is a dynamic organ that can adapt its structure to meet the demands of its biochemical and biophysical environment. Osteocytes form a sensory network throughout the tissue and orchestrate tissue adaptation via the release of soluble factors such as a sclerostin. Osteocyte physiology has traditionally been challenging to investigate due to the uniquely mineralized extracellular matrix (ECM) of bone leading to the development of osteocyte cell lines. Importantly, the most widely researched and utilized osteocyte cell line: the MLO-Y4, is limited by its inability to express sclerostin (Sost gene) in typical in-vitro culture. We theorised that culture in an environment closer to the in vivo osteocyte environment could impact on Sost expression. Therefore, this study investigated the role of composition and dimensionality in directing Sost expression in MLO-Y4 cells using collagen-based ECM analogues. A significant outcome of this study is that MLO-Y4 cells, when cultured on a hydroxyapatite (HA)-containing two-dimensional (2D) film analogue, expressed Sost. Moreover, three-dimensional (3D) culture within HA-containing collagen scaffolds significantly enhanced Sost expression, demonstrating the impact of ECM composition and dimensionality on MLO-Y4 behaviour. Importantly, in this bone mimetic ECM environment, Sost expression was found to be comparable to physiological levels. Lastly, MLO-Y4 cells cultured in these novel conditions responded accordingly to fluid flow stimulation with a decrease in expression. This study therefore presents a novel culture system for the MLO-Y4 osteocyte cell line, ensuring the expression of an important osteocyte specific gene, Sost, overcoming a major limitation of this model.

Download Full-text

Behaviour of Titanium Dioxide Particles in Artificial Body Fluids and Human Blood Plasma

International Journal of Molecular Sciences ◽

10.3390/ijms221910614 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10614

Author(s):

Eva Korábková ◽

Věra Kašpárková ◽

Daniela Jasenská ◽

Dita Moricová ◽

Eliška Daďová ◽

...

Keyword(s):

Blood Plasma ◽

Human Blood ◽

Body Fluids ◽

Human Blood Plasma ◽

Tio2 Particles ◽

Commercial Mixture ◽

Increased Risk ◽

The Impact

The growing application of materials containing TiO2 particles has led to an increased risk of human exposure, while a gap in knowledge about the possible adverse effects of TiO2 still exists. In this work, TiO2 particles of rutile, anatase, and their commercial mixture were exposed to various environments, including simulated gastric fluids and human blood plasma (both representing in vivo conditions), and media used in in vitro experiments. Simulated body fluids of different compositions, ionic strengths, and pH were used, and the impact of the absence or presence of chosen enzymes was investigated. The physicochemical properties and agglomeration of TiO2 in these media were determined. The time dependent agglomeration of TiO2 related to the type of TiO2, and mainly to the type and composition of the environment that was observed. The presence of enzymes either prevented or promoted TiO2 agglomeration. TiO2 was also observed to exhibit concentration-dependent cytotoxicity. This knowledge about TiO2 behavior in all the abovementioned environments is critical when TiO2 safety is considered, especially with respect to the significant impact of the presence of proteins and size-related cytotoxicity.

Download Full-text

Characterization of bioactive substances MHGF-68 on tumour cell lines with LiveFlow In Vitro Technology

European Pharmaceutical Journal ◽

10.2478/afpuc-2020-0021 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

R. Hodoši ◽

E. Nováková ◽

K. Macková ◽

M. Molitorisová ◽

M. Šupolíková

Keyword(s):

Cell Line ◽

Cell Morphology ◽

Tumour Cell ◽

Tumour Cell Line ◽

Bioactive Substances ◽

Dynamic Cultivation ◽

The Impact ◽

Cells Cultured

Abstract As part of experimental research, growth factor-like substances associated with MHV-68, named MHGF-68, were discovered in our laboratory. MHGF-68 activity was manifested by the ability to alter cell morphology, that is, normal phenotype to transformed, resp. suppresses the transformed phenotype of tumour cells. The aim of the experiments was to monitor the effect of MHGF-68 on the change of the cell actin cytoskeleton in the tumour cell line Hepa1c1c7, as well as the normal cell line NIH3T3, and compare conventional stationary cultivation and dynamic cultivation conditions using a LiveFlow system (In Vitro Technologies). LiveFlow is an advanced system to test the impact of different compounds on the cell cultures, which allows simulation of in vivo conditions thanks to continuous flow of cultivation medium. MHGF-68 was prepared with the infection of BHK-21 cells with MHV-68 virus under non-permissive conditions (41°C). After dynamic cultivation with MHGF-68, we observed changes in morphology on Hepa1c1c7 cells. In cells cultured in a dynamic environment, we observed more pronounced changes in cell morphology in comparison with cells cultured statically. We observed no changes in the cytoskeletal structures in the NIH 3T3 cell line affected by MHGF-68 in both types of cultivation. The advantage of LiveFlow in comparison to in vivo testing is that the experiments performed in this system are less time and money consuming. Dynamic cultivation in the LiveFlow system is suitable for optimizing experiments before testing substances in vivo.

Download Full-text

Complement activation induces dysregulation of angiogenic factors and causes fetal rejection and growth restriction

Journal of Experimental Medicine ◽

10.1084/jem.20061022 ◽

2006 ◽

Vol 203 (9) ◽

pp. 2165-2175 ◽

Cited By ~ 326

Author(s):

Guillermina Girardi ◽

Dmitry Yarilin ◽

Joshua M. Thurman ◽

V. Michael Holers ◽

Jane E. Salmon

Keyword(s):

Complement Activation ◽

Angiogenic Factors ◽

Growth Restriction ◽

Angiogenic Factor ◽

Vegf Receptor ◽

Placental Development ◽

Triggered Release ◽

Placental Dysfunction

Immune mechanisms have been implicated in placental dysfunction in patients with recurrent miscarriages and intrauterine growth restriction (IUGR), but the mediators are undefined. Here we show that complement activation, particularly C5a, is a required intermediary event in the pathogenesis of placental and fetal injury in an antibody-independent mouse model of spontaneous miscarriage and IUGR, and that complement activation causes dysregulation of the angiogenic factors required for normal placental development. Pregnancies complicated by miscarriage or growth restriction were characterized by inflammatory infiltrates in placentas, functional deficiency of free vascular endothelial growth factor (VEGF), elevated levels of soluble VEGF receptor 1 (sVEGFR-1, also known as sFlt-1; a potent anti-angiogenic molecule), and defective placental development. Inhibition of complement activation in vivo blocked the increase in sVEGFR-1 and rescued pregnancies. In vitro stimulation of monocytes with products of the complement cascade directly triggered release of sVEGFR-1, which sequesters VEGF. These studies provide the first evidence linking the complement system to angiogenic factor imbalance associated with placental dysfunction, and identify a new effector of immune-triggered pregnancy complications.

Download Full-text

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

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00093.2015 ◽

2015 ◽

Vol 309 (4) ◽

pp. E357-E369 ◽

Cited By ~ 11

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.

Download Full-text

The Role of the Adhesion Receptor CD146 and Its Soluble Form in Human Embryo Implantation and Pregnancy

Frontiers in Immunology ◽

10.3389/fimmu.2021.711394 ◽

2021 ◽

Vol 12 ◽

Author(s):

Sylvie Bouvier ◽

Elise Kaspi ◽

Ahmad Joshkon ◽

Odile Paulmyer-Lacroix ◽

Marie-Dominique Piercecchi-Marti ◽

...

Keyword(s):

Vascular System ◽

Embryo Implantation ◽

Angiogenic Factor ◽

Soluble Form ◽

Migration And Invasion ◽

Placental Development ◽

Extravillous Trophoblasts

CD146 is an adhesion molecule essentially located in the vascular system, which has been described to play an important role in angiogenesis. A soluble form of CD146, called sCD146, is detected in the bloodstream and is known as an angiogenic factor. During placental development, CD146 is selectively expressed in extravillous trophoblasts. A growing body of evidence shows that CD146 and, in particular, sCD146, regulate extravillous trophoblasts migration and invasion both in vitro and in vivo. Hereby, we review expression and functions of CD146/sCD146 in the obstetrical field, mainly in pregnancy and in embryo implantation. We emphasized the relevance of quantifying sCD146 in the plasma of pregnant women or in embryo supernatant in the case of in vitro fertilization (IVF) to predict pathological pregnancy such as preeclampsia or implantation defect. This review will also shed light on some major results that led us to define CD146/sCD146 as a biomarker of placental development and paves the way toward identification of new therapeutic targets during implantation and pregnancy.

Download Full-text