Wnt7a Decreases Brain Endothelial Barrier Function Via β-Catenin Activation

The blood-brain barrier consists of tightly connected endothelial cells protecting the brain’s microenvironment from the periphery. These endothelial cells are characterized by specific tight junction proteins such as Claudin-5 and Occludin, forming the endothelial barrier. Disrupting these cells might lead to blood-brain barrier dysfunction. The Wnt/β-catenin signaling pathway can regulate the expression of these tight junction proteins and subsequent barrier permeability. The aim of this study was to investigate the in vitro effects of Wnt7a mediated β-catenin signaling on endothelial barrier integrity. Mouse brain endothelial cells, bEnd.3, were treated with recombinant Wnt7a protein or XAV939, a selective inhibitor of Wnt/β-catenin mediated transcription to modulate the Wnt signaling pathway. The involvement of Wnt/HIF1α signaling was investigated by inhibiting Hif1α signaling with Hif1α siRNA. Wnt7a stimulation led to activation and nuclear translocation of β-catenin, which was inhibited by XAV939. Wnt7a stimulation decreased Claudin-5 expression mediated by β-catenin and decreased endothelial barrier formation. Wnt7a increased Hif1α and Vegfa expression mediated by β-catenin. However, Hif1α signaling pathway did not regulate tight junction proteins Claudin-5 and Occludin. Our data suggest that Wnt7a stimulation leads to a decrease in tight junction proteins mediated by the nuclear translocation of β-catenin, which hampers proper endothelial barrier formation. This process might be crucial in initiating endothelial cell proliferation and angiogenesis. Although HIF1α did not modulate the expression of tight junction proteins, it might play a role in brain angiogenesis and underlie pathogenic mechanisms in Wnt/HIF1α signaling in diseases such as cerebral small vessel disease

Investigation of the effects of phthalates on in vitro thyroid models with RNA-Seq and ATAC-Seq

IntroductionPhthalates are a class of endocrine-disrupting chemicals that have been shown to negatively correlate with thyroid hormone serum levels in humans and to cause a state of hyperactivity in the thyroid. However, their mechanism of action is not well described at the molecular level.MethodsWe analyzed the response of mouse thyroid organoids to the exposure to a biologically relevant dose range of the phthalates bis(2-ethylhexyl) phthalate (DEHP), di-iso-decylphthalate (DIDP), di-iso-nonylphthalate (DINP), and di-n-octylphthalate (DnOP) for 24 h and simultaneously analyzed mRNA and miRNA expression via RNA sequencing. Using the expression data, we performed differential expression and gene set enrichment analysis. We also exposed the human thyroid follicular epithelial cell line Nthy-ori 3-1 to 1 µM of DEHP or DINP for 5 days and analyzed changes in chromatin accessibility via ATAC-Seq.ResultsDose-series analysis showed how the expression of several genes increased or decreased at the highest dose tested. As expected with the low dosing scheme, the compounds induced a modest response on the transcriptome, as we identified changes in only mmu-miR-143-3p after DINP treatment and very few differentially expressed genes. No effect was observed on thyroid markers. Ing5, a component of histones H3 and H4 acetylation complexes, was consistently upregulated in three out of four conditions compared to control, and we observed a partial overlap among the genes differentially expressed by the treatments. Gene set enrichment analysis showed enrichment in the treatment samples of the fatty acid metabolism pathway and in the control of pathways related to the receptor signalling and extracellular matrix organization. ATAC-Seq analysis showed a general increase in accessibility compared to the control, however we did not identify significant changes in accessibility in the identified regions.DiscussionWith this work, we showed that despite having only a few differentially expressed genes, diverse analysis methods could be applied to retrieve relevant information on phthalates, showing the potential of in vitro thyroid-relevant systems for the analysis of endocrine disruptors

Hypoxic oligodendrocyte precursor cell-derived VEGFA is associated with blood–brain barrier impairment

Abstract Cerebral small vessel disease is characterised by decreased cerebral blood flow and blood–brain barrier impairments which play a key role in the development of white matter lesions. We hypothesised that cerebral hypoperfusion causes local hypoxia, affecting oligodendrocyte precursor cell—endothelial cell signalling leading to blood–brain barrier dysfunction as an early mechanism for the development of white matter lesions. Bilateral carotid artery stenosis was used as a mouse model for cerebral hypoperfusion. Pimonidazole, a hypoxic cell marker, was injected prior to humane sacrifice at day 7. Myelin content, vascular density, blood–brain barrier leakages, and hypoxic cell density were quantified. Primary mouse oligodendrocyte precursor cells were exposed to hypoxia and RNA sequencing was performed. Vegfa gene expression and protein secretion was examined in an oligodendrocyte precursor cell line exposed to hypoxia. Additionally, human blood plasma VEGFA levels were measured and correlated to blood–brain barrier permeability in normal-appearing white matter and white matter lesions of cerebral small vessel disease patients and controls. Cerebral blood flow was reduced in the stenosis mice, with an increase in hypoxic cell number and blood–brain barrier leakages in the cortical areas but no changes in myelin content or vascular density. Vegfa upregulation was identified in hypoxic oligodendrocyte precursor cells, which was mediated via Hif1α and Epas1. In humans, VEGFA plasma levels were increased in patients versus controls. VEGFA plasma levels were associated with increased blood–brain barrier permeability in normal appearing white matter of patients. Cerebral hypoperfusion mediates hypoxia induced VEGFA expression in oligodendrocyte precursor cells through Hif1α/Epas1 signalling. VEGFA could in turn increase BBB permeability. In humans, increased VEGFA plasma levels in cerebral small vessel disease patients were associated with increased blood–brain barrier permeability in the normal appearing white matter. Our results support a role of VEGFA expression in cerebral hypoperfusion as seen in cerebral small vessel disease

diXa: a data infrastructure for chemical safety assessment

Motivation: The field of toxicogenomics (the application of ‘-omics' technologies to risk assessment of compound toxicities) has expanded in the last decade, partly driven by new legislation, aimed at reducing animal testing in chemical risk assessment but mainly as a result of a paradigm change in toxicology towards the use and integration of genome wide data. Many research groups worldwide have generated large amounts of such toxicogenomics data. However, there is no centralized repository for archiving and making these data and associated tools for their analysis easily available. Results: The Data Infrastructure for Chemical Safety Assessment (diXa) is a robust and sustainable infrastructure storing toxicogenomics data. A central data warehouse is connected to a portal with links to chemical information and molecular and phenotype data. diXa is publicly available through a user-friendly web interface. New data can be readily deposited into diXa using guidelines and templates available online. Analysis descriptions and tools for interrogating the data are available via the diXa portal. Availability and implementation: http://www.dixa-fp7.eu Contact: [email protected]; [email protected] Supplementary information: Supplementary data are available at Bioinformatics onlin

Pharmacological inhibition of 17β-hydroxysteroid dehydrogenase impairs human endometrial cancer growth in an orthotopic xenograft mouse model

Endometrial cancer (EC) is the most common gynaecological tumor in developed countries and its incidence is increasing. Approximately 80% of newly diagnosed EC cases are estrogen-dependent. Type 1 17β-hydroxysteroid dehydrogenase (17β-HSD-1) is the enzyme that catalyzes the final step in estrogen biosynthesis by reducing the weak estrogen estrone (E1) to the potent estrogen 17β-estradiol (E2), and previous studies showed that this enzyme is implicated in the intratumoral E2 generation in EC. In the present study we employed a recently developed orthotopic and estrogen-dependent xenograft mouse model of EC to show that pharmacological in-hibition of the 17β-HSD-1 enzyme inhibits disease development. Tumors were induced in one uterine horn of athymic nude mice by  intrauterine injection of  the  well-differentiated human endometrial adenocarcinoma Ishikawa cell line, modified to express human 17β-HSD-1 in levels comparable to EC, and the luciferase and green fluorescent protein reporter genes. Controlled estrogen exposure in ovariectomized mice was achieved using subcutaneous MedRod implants that released either the low active estrone (E1) precursor or vehicle. A subgroup of E1 supplemented mice received daily oral gavage of FP4643, a well-characterized 17β-HSD-1 in-hibitor. Bioluminescence imaging (BLI) was used to measure tumor growth non-invasively. At sacrifice, mice receiving E1  and  treated with the  FP4643 inhibitor showed a  significant reduction in  tumor growth by approximately 65% compared to mice receiving E1. Tumors exhibited metastatic spread to the peritoneum, to the  lymphovascular space (LVI), and  to  the  thoracic cavity. Metastatic spread and  LVI  invasion were both significantly reduced in the inhibitor-treated group. Transcriptional profiling of tumors indicated that FP4643 treatment reduced the oncogenic potential at the mRNA level. In conclusion, we show that 17β-HSD-1 inhibition represents a promising novel endocrine treatment for EC.   </div

Versuche uber Immunisierung von dem Konjunktivalsack aus mit Berucksichtigung des Einflusses der Ultraviolettbestrahlung auf den Immunisierungsvorgang

1) In Vorversuchen hat der Verfasser eingehend einerseits den direkten Einfluss der Ultraviolettstrahlen auf die beabsichtigte Antigene, ihre Suspensions-bzw. Losungs-medien so wie auf die in Immunserum anwesenden Antikorper festgestellt, anderseits die lokale und allgemeine Einwirkung auf den Tierkorper der U. V. Bestrahlung des Kaninchenauges sichergestellt. Im allgemeinen hat die U. V. Bestrahlung auf lebende Bakterien verschiedener Rassen bakterizide Wirkung entfaltet. Die Wirkungskraft war bei verschiedenen Bakterienarten, in verschiedenen Suspensionsmedien und je nach der Bestrahlungsweise verschieden. Auf das Auge selbst hat die U. V. Bestrahlung, vorausgesetzt, dass sie zweck-massig durchgefuhrt wird, keinen ublen Einfluss. Die Keime des Konjunktivalsackes nehmen als Folge der Bestrahlung an ihrer Zahl ab. Als allgemeiner Einfluss auf den Tierkorper kann der Aufstieg der Korpertem-peratur nach der Bestrahlung und die Korpergewichtzunahme erwahnt werden. Uber-massiges Bestrahlen fuhrt zur Gewichtsabnahme. Im Gegenteil zu der Wirkung im immuisierten Korper verliert das direkt bestr-ahlte Immunserum an seinem Antikorpergehalt. 2) Durch Agglutinationsproben wurde der Immunisierungsvorgang verfolgt und die Moglichkeit der Immunisierung vom Konjumktivalsack aus festgestellt. Es kamen die folgenden Bakterien in Frage; B. typhi B. prodigiosus, Staphylokokken, Gonoko-kken, Pneumokokken, V. Metschnikoffi. Als Aufschwemmungsmedium der bakteriellen Antigene wurde Kochsalzlosung, Dionin-, NaHCO3-, und verdunnte HCl- losung gebraucht. Diese Immunisierungsversuche wurden teilweise mit, teilweise ohne U. V. Bestrahlung durchgefuhrt, wodurch die Tatsache festgestellt wurde, dass die Bestr-ahlung auf die Agglutininbildung einen gunstigen Einfluss ausubt. 3) Ahnlich wie mit den verschiedenen Bakterien wurden auch Immunisierungs-versuche mit Diphterietoxin angestellt. Es ist zwar gelungen im Blute der auf die in Frage stehende Weise behandelten Versuchstiere das spezifische Antitoxin nachzuweisen, aber es lag kein Beweis a

Contribution to the knowledge of polar overdominance in sheep callipyge locus

THÈME DE RECHERCHE :Le phénotype callipyge est une hypertrophie musculaire généralisée post-natale décrite chez le mouton (introduit dans le Chapitre 1). Son mode de transmission non mendélien, qualifié de surdominance polaire, est unique : seuls les hétérozygotes ayant reçu la mutation callipyge (CLPG) de leur père expriment le phénotype. La mutation CLPG, localisée dans un domaine soumis à l'empreinte parentale, est une mutation ponctuelle (SNP) détruisant un élément qui contrôle, en cis, le taux d’expression musculaire des gènes voisins. L'hypertrophie musculaire, sans doute causée par l'expression ectopique de la protéine DLK1 chez les animaux +mat/Cpat, n'est pas observée chez les animaux Cmat/Cpat dont l'allèle maternel apparaît trans-inactiver la traduction du messager DLK1. Cette thèse a pour objectif d'approfondir notre compréhension de la surdominance polaire, aussi bien au travers de l'étude des mécanismes impliqués dans l'effet de contrôle à longue distance en cis de la mutation que de la caractérisation des inhibitions en trans induites par les transcrits maternels sur les messagers paternels. RÉSULTATS :L'étude de l'effet en cis (Chapitre 2) a démontré que la mutation CLPG se différenciait de l'allèle sauvage par au moins trois marques épigénétiques distinctes : (i) l'hypométhylation de l'ADN à proximité du SNPCLPG, (ii) la création d'un site d'hypersensibilité à la DNase, et (iii) l'activation de la transcription bidirectionnelle de la région autour de la mutation. En démontrant que la mutation inactivait bel et bien un élément de contrôle à longue distance, ces données nous ont permis d'élaborer un modèle plus précis de la surdominance polaire. L'étude de la trans-inhibition des transcrits protéiques à expression paternelle par les transcrits non codants maternels (Chapitres 3 et 4) se fondait sur l'hypothèse d'une implication des nombreux microARNs (miRNAs) du domaine DLK1-GTL2. Nous avons ainsi pu montrer que les cinq miRNAs abrités par anti-PEG11 étaient capables, par leur parfaite complémentarité de séquence, de cliver le messager PEG11 (Chapitre 3). Bien que le rôle de la protéine PEG11 dans le phénotype callipyge soit inconnu, cette étude a néanmoins démontré l'existence d'une trans-inhibition entre les deux allèles du locus, tout en identifiant le premier cas de dégradation miRNA/cible impliquant des gènes soumis à l'empreinte chez les mammifères. L'étude de la trans-inhibition appliquée au messager DLK1 (Chapitre 4) a quant à elle nécessité la génération par séquençage haut débit d'un catalogue exhaustif des miRNAs du muscle squelettique ovin. Ce travail nous a permis de démontrer que les miRNAs du domaine DLK1-GTL2 étaient bien exprimés maternellement et eux aussi soumis à l'effet en cis de la mutation. Si aucun miRNA capable d'inhiber le messager DLK1 n'a été identifié sans ambiguïté, nos analyses d'affinité ont toutefois révélé un effet significatif de miR-376c sur sa 3'UTR, ainsi que de l'ensemble des miRNAs du locus DLK1-GTL2 sur sa région codante. Notons que nous avons également considéré comme potentiels candidats à la trans-inhibition les snoRNAs du locus, de même que les miRNAs du locus soumis à l'édition ARN.CONCLUSIONS ET PERSPECTIVES :À l'issue de cette thèse, nous avons donc contribué à la compréhension du phénomène de surdominance polaire au locus DLK1-GTL2 du mouton callipyge. Si de nombreuses questions restent en suspend, elles devraient néanmoins trouver réponse grâce aux nouveaux outils d'analyse en cours de développement. Ainsi, deux lignées de souris transgéniques développées dans notre laboratoire, respectivement porteuses de la mutation CLPG ou surexprimant PEG11 dans le muscle, éclaireront les mécanismes moléculaires de l'effet en cis de la mutation ainsi que le rôle de PEG11 dans l'établissement du phénotype callipyge. Par ailleurs, pour confirmer in vivo l'effet inhibiteur sur DLK1 des miRNAs identifiés dans notre étude, les progrès apportés par le séquençage haut débit (notamment le HITS-CLIP) et la transfection en modèle cellulaire se révéleront sans doute d'une grande aide. / RESEARCH OVERVIEW:The callipyge phenotype is a generalized muscular hypertrophy described in sheep (see Chapter 1). It features a non-mendelian mode of inheritance termed polar overdominance: only heterozygous animals having inherited the mutation from their father display the phenotype. The callipyge mutation (CLPG), which falls in an imprinted domain, is a single-nucleotide polymorphism (SNP) that disrupts a putative long-range control element affecting, in cis, the expression level of neighboring imprinted genes in skeletal muscle. The callipyge phenotype is thought to be caused by ectopic expression of DLK1 protein in +mat/Cpat animals. In contrast, Cmat/Cpat animals exhibit a wild-type phenotype that is certainly due to a trans-inhibition from the maternal allele on DLK1 translation. The objective of our thesis was to improve our knowledge of polar overdominance, both by studying mechanisms of long-range cis regulation and by characterizing the trans effect of maternal noncoding transcripts on the paternally-expressed messenger RNAs. RESULTS:Our study of the cis effect (Chapter 2) showed that the CLPG mutation differs from the wild-type allele by at least three distinct epigenetic marks: (i) DNA hypomethylation in the vicinity of the SNPCLPG, (ii) creation of a DNase-hypersensitive site, and (iii) activation of a bidirectional transcription start site centered on the mutation. Altogether, our data provided strong evidence for the SNPCLPG inactivating a long-range control element and allowed us to refine our model of polar overdominance. Our working hypothesis for the trans-inhibition of paternally-expressed genes by maternal noncoding transcripts (Chapters 3 and 4) involved microRNAs (miRNAs) from the DLK1-GTL2 domain. In this respect, we showed that five miRNAs from anti-PEG11 were able to cleave PEG11 transcript, owing to perfect sequence complementarity (Chapter 3). This study was the first demonstration of miRNA-mediated RNAi involving imprinted genes in mammals. Furthermore, it allowed us to confirm the existence of a trans-inhibition between both alleles in the domain, albeit the role of PEG11 protein in the callipyge phenotype is still unknown. To study the trans-inhibition of DLK1 messenger (Chapter 4), we used high-throughput sequencing to build an exhaustive catalogue of skeletal muscle-specific miRNAs in sheep. Our analyses showed that miRNAs from the DLK1-GTL2 domain are maternally expressed and affected by the cis effect of the CLPG mutation. Even if we could not find miRNAs unambiguously able to repress DLK1 transcript, affinity analyses revealed a significant effect on its 3' UTR for miR-376c, as well as on its coding region for all miRNAs considered as team. Of note, we also investigated snoRNAs and miRNAs subjected to RNA editing in the DLK1-GTL2 locus.CONCLUSIONS AND PERSPECTIVES:During the course of this research, we contributed to improve the understanding of polar overdominance in the sheep DLK1-GTL2 locus. Although many questions remain, most will eventually be answered thanks to upcoming analysis tools. Hence, our lab has already generated two transgenic mouse lines, either carrying the CLPG mutation or over-expressing PEG11 in skeletal muscle. These mice should respectively shed light on the molecular mechanisms underlying the cis effect of the CLPG mutation and on the role of PEG11 in the callipyge phenotype. Finally, to confirm inhibiting effects of miRNAs identified in our study, technological improvements granted by high-throughput sequencing (such as HITS-CLIP) and miRNAs transfection in cell model systems will both prove to be very useful