Influence of 4-methylumbelliferone, a hyaluronan-synthase inhibitor, on atherosclerotic vessel alterations in apoE-deficient mice

Hyaluronsäure (HA) ist ein bedeutender Bestandteil atherosklerotischer Läsionen, vermittelt neointimale Expansion und fördert die Migration und Proliferation glatter Muskelzellen. Andererseits ist Hyaluronsäure ein integraler Bestandteil der vasoprotektiven endothelialen Glykokalyx. In der vorliegenden Arbeit wurden zum ersten Mal die Effekte einer chronischen pharmakologischen Inhibition der HA-Synthese auf Atherosklerose und Gefäßfunktion untersucht. ApoE-defiziente Mäuse, die ein fett- und cholesterinreiches Futter erhielten, wurden ab einem Alter von vier Wochen oral mit 4 Methylumbelliferon (4-MU), einem Inhibitor der HA-Synthese, behandelt. Anschließend wurde der Plaquebesatz der Aorta und die Plaquefläche des Aortenursprungs im Alter von 8, 15 und 25 Wochen bestimmt. Bei dem 25 Wochen Untersuchungszeitpunkt konnte eine deutliche Steigerung der Atherosklerose festgestellt werden, die mit einer Anhäufung von mac2-positiven Makrophagen in den atherosklerotischen Läsionen einherging. Weiterhin konnte gezeigt werden, dass die Acetylcholin-abhängige Vasorelaxation von isolierten Aortenringen verschlechtert, und der systolische Blutdruck unter Einfluss von 4-Methylumbelliferon erhöht war. Mit Hilfe des Modells der photochemisch induzierten Thrombose konnte eine verstärkte Thromboseneigung unter Einfluss von 4-MU gezeigt werden. Der pro-thrombotische Status wurde weder durch eine erhöhte Plättchenaktivität, bestimmt durch CD62P Expression, noch durch eine veränderte Thrombinaktivität, bestimmt durch die Messung des endogenen Thrombinpotentials, ausgelöst. Der Effekt von 4-MU auf die Volumenexpansion atherosklerotischer Plaques war spezifisch für die Atherosklerose da 4-MU auf die Neointimahyperplasie, ausgelöst durch die Ligatur der linken A. carotis, keine Effekte hatte. Elektronenmikroskopische Aufnahmen von myokardialen Kapillaren zeigten eine Schädigung der endothelialen Glykokalyx unter dem Einfluss von 4-MU. Zusammenfassend konnte gezeigt werden, dass die systemische Inhibition der Hyaluronsäure-Synthese zu einer beschleunigten Atherosklerose führt. Als mögliche Ursache wird der Verlust der vasoprotektiven endothelialen Glykokalyx durch 4 Methylumbelliferon angenommen.Hyaluronan (HA) is thought to mediate neointimal expansion and to facilitate smooth muscle cell migration and proliferation. On the other hand HA is an integral component of the endothelial glycocalyx mediating vasoprotection. The present study examines for the first time the effects of chronic pharmacologic inhibition of HA synthesis, on atherosclerosis and vascular function. ApoE-deficient mice on a high-fat and cholesterol-rich Western-diet were treated orally with 4-methylumbelliferone (4-MU), an inhibitor of HA-synthesis, beginning at four weeks of age. Subsequently, aortic atherosclerotic plaque burden and plaque size at the aortic root level were determined at 8, 15 and 25 weeks of age. Atherosclerosis was markedly increased at 25 weeks and accumulation of mac2-positive macrophages was increased in the lesions. Furthermore, acetylcholine dependent relaxation of aortic rings was decreased and mean arterial blood pressure was increased in response to 4-MU. The model of photochemically induced thrombosis revealed a pro-thrombotic state under 4-MU treatment, that was not due to increased platelet activation as determined by CD62P expression and did not reflect increased thrombin activity as monitored by the endogenous thrombin potential. Furthermore, the lesion promoting effect of 4-MU was specific for atherosclerosis since 4-MU did not accelerate neointimal hyperplasia in response to ligation of the left carotid artery. Electron microscopy of myocardial capillaries revealed a severely damaged endothelial glycocalyx after 4-MU treatment. The data suggest that systemic inhibition of HA synthesis by 4-MU accelerates atherosclerosis possibly due to ablation of the protective function of the endothelial glycocalyx

Clinical sequencing identifies potential actionable alterations in a high rate of urachal and primary bladder adenocarcinomas.

OBJECTIVE Administration of targeted therapies provides a promising treatment strategy for urachal adenocarcinoma (UrC) or primary bladder adenocarcinoma (PBAC); however, the selection of appropriate drugs remains difficult. Here, we aimed to establish a routine compatible methodological pipeline for the identification of the most important therapeutic targets and potentially effective drugs for UrC and PBAC. METHODS Next-generation sequencing, using a 161 cancer driver gene panel, was performed on 41 UrC and 13 PBAC samples. Clinically relevant alterations were filtered, and therapeutic interpretation was performed by in silico evaluation of drug-gene interactions. RESULTS After data processing, 45/54 samples passed the quality control. Sequencing analysis revealed 191 pathogenic mutations in 68 genes. The most frequent gain-of-function mutations in UrC were found in KRAS (33%), and MYC (15%), while in PBAC KRAS (25%), MYC (25%), FLT3 (17%) and TERT (17%) were recurrently affected. The most frequently affected pathways were the cell cycle regulation, and the DNA damage control pathway. Actionable mutations with at least one available approved drug were identified in 31/33 (94%) UrC and 8/12 (67%) PBAC patients. CONCLUSIONS In this study, we developed a data-processing pipeline for the detection and therapeutic interpretation of genetic alterations in two rare cancers. Our analyses revealed actionable mutations in a high rate of cases, suggesting that this approach is a potentially feasible strategy for both UrC and PBAC treatments

Analysis of Blood Stem Cell Activity and Cystatin Gene Expression in a Mouse Model Presenting a Chromosomal Deletion Encompassing Csta and Stfa2l1

The cystatin protein superfamily is characterized by the presence of conserved sequences that display cysteine protease inhibitory activity (e.g., towards cathepsins). Type 1 and 2 cystatins are encoded by 25 genes of which 23 are grouped in 2 clusters localized on mouse chromosomes 16 and 2. The expression and essential roles of most of these genes in mouse development and hematopoiesis remain poorly characterized. In this study, we describe a set of quantitative real-time PCR assays and a global expression profile of cystatin genes in normal mouse tissues. Benefiting from our collection of DelES embryonic stem cell clones harboring large chromosomal deletions (to be reported elsewhere), we selected a clone in which a 95-kb region of chromosome 16 is missing (Del16qB3Δ/+). In this particular clone, 2 cystatin genes, namely Csta and Stfa2l1 are absent along with 2 other genes (Fam162a, Ccdc58) and associated intergenic regions. From this line, we established a new homozygous mutant mouse model (Del16qB3Δ/16qB3Δ) to assess the in vivo biological functions of the 2 deleted cystatins. Stfa2l1 gene expression is high in wild-type fetal liver, bone marrow, and spleen, while Csta is ubiquitously expressed. Homozygous Del16qB3Δ/16qB3Δ animals are phenotypically normal, fertile, and not overtly susceptible to spontaneous or irradiation-induced tumor formation. The hematopoietic stem and progenitor cell activity in these mutant mice are also normal. Interestingly, quantitative real-time PCR expression profiling reveals a marked increase in the expression levels of Stfa2l1/Csta phylogenetically-related genes (Stfa1, Stfa2, and Stfa3) in Del16qB3Δ/16qB3Δ hematopoietic tissues, suggesting that these candidate genes might be contributing to compensatory mechanisms. Overall, this study presents an optimized approach to globally monitor cystatin gene expression as well as a new mouse model deficient in Stfa2l1/Csta genes, expanding the available tools to dissect cystatin roles under normal and pathological conditions

Genome-Wide Interrogation of Mammalian Stem Cell Fate Determinants by Nested Chromosome Deletions

Understanding the function of important DNA elements in mammalian stem cell genomes would be enhanced by the availability of deletion collections in which segmental haploidies are precisely characterized. Using a modified Cre-loxP–based system, we now report the creation and characterization of a collection of ∼1,300 independent embryonic stem cell (ESC) clones enriched for nested chromosomal deletions. Mapping experiments indicate that this collection spans over 25% of the mouse genome with good representative coverage of protein-coding genes, regulatory RNAs, and other non-coding sequences. This collection of clones was screened for in vitro defects in differentiation of ESC into embryoid bodies (EB). Several putative novel haploinsufficient regions, critical for EB development, were identified. Functional characterization of one of these regions, through BAC complementation, identified the ribosomal gene Rps14 as a novel haploinsufficient determinant of embryoid body formation. This new library of chromosomal deletions in ESC (DelES: http://bioinfo.iric.ca/deles) will serve as a unique resource for elucidation of novel protein-coding and non-coding regulators of ESC activity

CMB-S4

We describe the stage 4 cosmic microwave background ground-based experiment CMB-S4

Optical spectra and microscopic structure of the oxidized Si(100) surface: Combined in situ optical experiments and first principles calculations

International audienceWe have investigated the first stages of the room-temperature oxidation of the Si100 surface combining experimental surface optical spectra with the results of ab initio calculations. High-resolution reflectance anisotropy spectra RAS and surface differential reflectance spectra SDRS have been measured for the clean surfaces and various exposures up to 183 L, which have been compared with calculated RAS and SDRS in the independent-particle approximation. Our results, yielding a consistent description of both RAS and SDRS, suggest the coexistence of different structural domains, whose weight changes smoothly with the oxygen exposure. The main oxidation mechanisms together with their occurrence versus coverage are discussed

Induced lactation with a dopamine antagonist in mares : different responses between ovariectomized and intacts mares

International audienc

Cyclooxygenase inhibitors repress vascular hyaluronan-synthesis in murine atherosclerosis and neointimal thickening

Marzoll A, Nagy N, Wördehoff L, et al. Cyclooxygenase inhibitors repress vascular hyaluronan-synthesis in murine atherosclerosis and neointimal thickening. Journal of Cellular and Molecular Medicine. 2009;13(9b):3713-3719.Hyaluronan (HA) is a key molecule of the extracellular matrix that is thought to be critically involved in both atherosclerosis and restenosis. Recently, it has been demonstrated that the cyclooxygenase (COX) products, prostacyclin and prostaglandin E2, induce HA synthesis in vitro by transcriptional up-regulation of HA-synthase 2 (HAS2) and HAS1. The relative roles in atherosclerotic and restenotic artery disease of tissue specifically expressed COX-1 and COX-2 are still under debate. Thus, the present study aimed to investigate the effect of COX isoform inhibition on HA-accumulation and regulation of HAS isoform expression in two models of pathologic artery remodelling in vivo. Firstly, ApoE-deficient mice were treated with a prototypic isoform non-selective inhibitor, indomethacin or with a prototypic COX-2 selective inhibitor, rofecoxib, for 8 weeks. Aortic HAS mRNA expression and HA-accumulation in atherosclerotic aortic root lesions were analyzed. Secondly, neointimal hyperplasia was induced by carotid artery ligation in ApoE-deficient mice on a high fat diet and the effects of the COX inhibitors were determined after 4 weeks of treatment. Intimal HA-accumulation was markedly reduced in both models by indomethacin and rofecoxib. This coincided with a strong inhibition of HAS1 mRNA expression in both models and with decreased HAS2 mRNA in the aorta of ApoE-deficient mice. HAS3 was not affected. The repression of HA-accumulation by both COX-2 selective and non-selective COX inhibition implicates COX-2 in the regulation of HA synthesis via stimulation of HAS1 and HAS2 expression in vivo. Modulation of vascular HA-accumulation might play a role in chronic effects of COX inhibitors on the progression of atherosclerosis