Effects of GSK3 inhibitors on in vitro expansion and differentiation of human adipose-derived stem cells into adipocytes

<p>Abstract</p> <p>Background</p> <p>Multipotent stem cells exist within adipose tissue throughout life. An abnormal recruitment of these adipose precursor cells could participate to hyperplasia of adipose tissue observed in severe obesity or to hypoplasia of adipose tissue observed in lipodystrophy. Therefore, pharmacological molecules that control the pool of stem cells in adipose tissue are of great interest. Glycogen Synthase Kinase (GSK) 3 has been previously described as involved in differentiation of preadipose cells and might be a potential therapeutic target to modulate proliferation and differentiation of adipocyte precursors. However, the impact of GSK3 inhibition on human adipose-derived stem cells remained to be investigated. The aim of this study was to investigate GSK3 as a possible target for pharmacological inhibition of stem cell adipogenesis. To reach this goal, we studied the effects of pharmacological inhibitors of GSK3, i.e. lithium chloride (LiCl) and BIO on proliferation and adipocyte differentiation of multipotent stem cells derived from human adipose tissue.</p> <p>Results</p> <p>Our results showed that GSK3 inhibitors inhibited proliferation and clonogenicity of human stem cells, strongly suggesting that GSK3 inhibitors could be potent regulators of the pool of adipocyte precursors in adipose tissue. The impact of GSK3 inhibition on differentiation of hMADS cells was also investigated. Adipogenic and osteogenic differentiations were inhibited upon hMADS treatment with BIO. Whereas a chronic treatment was required to inhibit osteogenesis, a treatment that was strictly restricted to the early step of differentiation was sufficient to inhibit adipogenesis.</p> <p>Conclusion</p> <p>These results demonstrated the feasibility of a pharmacological approach to regulate adipose-derived stem cell function and that GSK3 could represent a potential target for controlling adipocyte precursor pool under conditions where fat tissue formation is impaired.</p

Membrane estrogen receptor-α contributes to female protection against high-fat diet-induced metabolic disorders

BackgroundEstrogen Receptor α (ERα) is a significant modulator of energy balance and lipid/glucose metabolisms. Beyond the classical nuclear actions of the receptor, rapid activation of intracellular signaling pathways is mediated by a sub-fraction of ERα localized to the plasma membrane, known as Membrane Initiated Steroid Signaling (MISS). However, whether membrane ERα is involved in the protective metabolic actions of endogenous estrogens in conditions of nutritional challenge, and thus contributes to sex differences in the susceptibility to metabolic diseases, remains to be clarified.MethodsMale and female C451A-ERα mice, harboring a point mutation which results in the abolition of membrane localization and MISS-related effects of the receptor, and their wild-type littermates (WT-ERα) were maintained on a normal chow diet (NCD) or fed a high-fat diet (HFD). Body weight gain, body composition and glucose tolerance were monitored. Insulin sensitivity and energy balance regulation were further investigated in HFD-fed female mice.ResultsC451A-ERα genotype had no influence on body weight gain, adipose tissue accumulation and glucose tolerance in NCD-fed mice of both sexes followed up to 7 months of age, nor male mice fed a HFD for 12 weeks. In contrast, compared to WT-ERα littermates, HFD-fed C451A-ERα female mice exhibited: 1) accelerated fat mass accumulation, liver steatosis and impaired glucose tolerance; 2) whole-body insulin resistance, assessed by hyperinsulinemic-euglycemic clamps, and altered insulin-induced signaling in skeletal muscle and liver; 3) significant decrease in energy expenditure associated with histological and functional abnormalities of brown adipose tissue and a defect in thermogenesis regulation in response to cold exposure.ConclusionBesides the well-characterized role of ERα nuclear actions, membrane-initiated ERα extra-nuclear signaling contributes to female, but not to male, protection against HFD-induced obesity and associated metabolic disorders in mouse

Reprogramming of endothelial gene expression by tamoxifen inhibits angiogenesis and ERα-negative tumor growth.

peer reviewedRationale: 17β-estradiol (E2) can directly promote the growth of ERα-negative cancer cells through activation of endothelial ERα in the tumor microenvironment, thereby increasing a normalized tumor angiogenesis. ERα acts as a transcription factor through its nuclear transcriptional AF-1 and AF-2 transactivation functions, but membrane ERα plays also an important role in endothelium. The present study aims to decipher the respective roles of these two pathways in ERα-negative tumor growth. Moreover, we delineate the actions of tamoxifen, a Selective Estrogen Receptor Modulator (SERM) in ERα-negative tumors growth and angiogenesis, since we recently demonstrated that tamoxifen impacts vasculature functions through complex modulation of ERα activity. Methods: ERα-negative B16K1 cancer cells were grafted into immunocompetent mice mutated for ERα-subfunctions and tumor growths were analyzed in these different models in response to E2 and/or tamoxifen treatment. Furthermore, RNA sequencings were analyzed in endothelial cells in response to these different treatments and validated by RT-qPCR and western blot. Results: We demonstrate that both nuclear and membrane ERα actions are required for the pro-tumoral effects of E2, while tamoxifen totally abrogates the E2-induced in vivo tumor growth, through inhibition of angiogenesis but promotion of vessel normalization. RNA sequencing indicates that tamoxifen inhibits the E2-induced genes, but also initiates a specific transcriptional program that especially regulates angiogenic genes and differentially regulates glycolysis, oxidative phosphorylation and inflammatory responses in endothelial cells. Conclusion: These findings provide evidence that tamoxifen specifically inhibits angiogenesis through a reprogramming of endothelial gene expression via regulation of some transcription factors, that could open new promising strategies to manage cancer therapies affecting the tumor microenvironment of ERα-negative tumors

L'utilisation des nouvelles technologies de l'information et de la communication dans la pratique professionnelle de l'avocat – État des lieux des possibilités offertes par l'outil technologique au cyberavocat

Master [120] en droit, Université catholique de Louvain, 201

Rôle et régulation du récepteur nucléaire orphelin REV-ERB a (voies d'interaction avec les récepteurs nucléaires PPARg et LXR)

Les récepteurs nucléaires sont des cibles pharmacologiques très étudiées, leur activation permettant de moduler de nombreux dysfonctionnements physiologiques. Le récepteur nucléaire Rev-erba, aujourd'hui reconnu pour son rôle dans la régulation de la rythmicité circadienne, est un récepteur nucléaire orphelin puisque à ce jour aucun ligand n'a été identifié. Rev-erba agit en réprimant la transcription de ses gènes cibles. L'objectif de ces travaux a été de préciser le rôle physiologique de Rev-erba. L'approche utilisée consiste à déterminer les voies d'interaction existant avec d'autres récepteurs nucléaires dont les modes d'action sont bien établis, le " Peroxisme Proliferator-Activated Receptor " (PPAR)g et les " Liver X Receptor " (LXR). Ces récepteurs jouent un rôle majeur dans le développement de l'athérosclérose et dans les anomalies métaboliques qui lui sont associées. Ils agissent en activant la transcription de leurs gènes cibles en se fixant sur des séquences d'ADN spécifiques dont certaines sont également reconnues par Rev-erba. Ainsi, nous avons montré que PPARg, un acteur clé de la différenciation adipocytaire, régule l'expression de Rev-erba dans les adipocytes. La surexpression de Rev-erba potentialise la différenciation adipocytaire induite par l'activation induite par l'activation de PPARg. Rev-erba a donc été identifié comme un nouveau gène cible de PPARg qui agirait en aval de ce dernier pour favoriser son action adipocytire. Parallèlement, dans les macrophages primaires humains, l'activation des LXR conduit à une augmentation de l'expression de Rev-erba. Bien que les LXR jouent un rôle majeur dans l'élimination du cholestérol des macrophages, Rev-erba ne régule pas l'homéostasie du cholestérol dans ces cellules. LXR étant impliqué dans la réponse inflammatoire et dans la réponse immune du macrophage, nous avons entrepris d'étudier le " cross-talk " des LXR et de Rev-erba dans ce contexte. Ainsi, nous avons montré que Rev-erba inhibe l'activation des LXR sur l'expression du " Toll-like receptor " (TLR)-4, le récepteur au lipopolysaccharide (LPS). L'identification d'un nouveau gène cible commun pour les LXR et Rev-erba répond au schéma préalablement décrit quant à l'interaction entre les voies de signalisation de Rev-erba avec d'autres récepteurs nucléaires tels que le " RAR-related orphan receptor " (ROR) et PPARa. Rev-erba apparaît donc come un lien moléculaire à l'origine de rétrocontrôles négatifs. De par son mode d'action, Rev-erba apparaît comme un intégrateur entre différentes voies de signalisation, notamment entre rythmicité circadienne, métabolisme et inflammation.Nuclear receptors (NR) function as ligand-dependent transcription factors that bind DNA and transduce physiological signals into gene regulation. Nucler receptors regulate gene expression involved in reproduction, development and metabolism. Recent genetic, pharmalogical and biochemical studies on members of the nuclear receptor superfamily (e.g. " Peroxisome Proliferator-Activated Receptors " (PPAR), " Liver X Receptors " (LXR)) have uncovered new signaling pathways that regulate metabolism, and provided new insights into the pathophysiology of diabetes, dyslipidemia, obesity, vascular inflammation. In addition to the ligand-activated Nrs, many members within this superfamily have no ligand, and are referred to as " orphan nuclear receptors ". Rev-erba (NR1D1) is an orphan member of the steroid/thyroid hormone receptor nuclear family, which acts as a repressor of genes transcription. The aim of this study was to investigate a possible cross-talk between these nuclear receptors. First, we identified Rev-erba as a new target gene for PPARg in the adipogenic cascade of transcription factors and as an important factor modulating adipocyte function, at least in part, by enhancing the adipogenic action of PPARg. In a second part of this work, we find that LXR activation by synthetic agonists induces Rev-erba mRNA expression in human primary macro^phages. In addition, the results reported here show that Rev-erba acts as negative regulator of LXR transactivation, on a new LXR target gene, the LPS receptor, TLR-4. Interestingly, LXR and Rev-erba bind to the same response element on the TLR-4 promoter. As such, Rev-erba inhibits transcriptional activation mediated by nuclear receptors, which directly regulate Rev-erba expression. To conclude, Rev-erba appears to drive transcriptional feedback loops of many nuclear receptors controlling physiological processes. Thus, the Rev-erba regulation pathway may converge to integrate metabolism, inflammation and circadian cycle conferring physiological flexibility.LILLE2-BU Santé-Recherche (593502101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Evaluation de la prise en charge antalgique lors de la vaccination chez les enfants de moins de 4 mois

CONTEXTE : La prise en charge de la douleur est devenue une préoccupation majeure ces dernières années. Les stimulations douloureuses sont présentes chez le nourrisson et peuvent parfois être anticipables, notamment lors des injections de vaccins. L'utilisation d'antalgiques lors des actes douloureux a montré son efficacité, mais ces moyens bien que connus ne sont pas toujours systématiques. L objectif de cette étude est d'évaluer le ressenti douloureux des moins de quatre mois lors de la réalisation des vaccins dans la région amiénoise pour permettre d'essayer de dégager une prise en charge antalgique efficace. MATERIELS ET METHODES : Une étude observationnelle d'une durée de six mois a été réalisée au CHU d'Amiens et dans les services de PMI de la Somme comportant deux parties: une première partie recueillant les habitudes de pratiques vaccinales des pédiatres de Picardie et une deuxième partie évaluant le ressenti douloureux en consultation lors des vaccins à l'aide du temps de pleurs et de la valeur du DAN au moment de l'injection en fonction du type d'antalgie reçue (solution sucrée, succion, crème anesthésiante ou rien) et du type de vaccin (Infanrix® ou Prévenar®). RESULTATS : 144 enfants ayant reçu deux injections et ayant un DAN < 4 ont été inclus dans cette étude. Aucune différence significative n'a été mise en évidence entre les différents antalgiques utilisés. Par contre les enfants installés dans les bras de leurs parents semblaient moins ressentir la douleur que sur la table d'examen lors du Prevenar® (valeur DAN p < 0,04). L'Infanrix® était toujours réalisé en premier et semblait être moins douloureux que le Prévenar®, car le temps de pleurs était significativement plus court (p < 0,001). En fin de consultation, tous les enfants avaient une valeur de DAN inférieure à 3 et ne nécessitaient pas d'antalgique supplémentaire. CONCLUSION : Nous n'avons pas pu dégager une prise en charge antalgique optimale devant l'absence de différence significative entre les différents groupes d'antalgiques. Par contre cette étude montre l'importance d'une préparation optimale au geste et d'une évaluation systématique du ressenti douloureux à chaque acte invasif.BACKGROUND: Strategies to improve pain management in children have been developed for the last years. Invasive procedures in children can often be anticipated. Vaccination is the most common iatrogenic procedure during childhood. Treatments can prevent pain but they are not enough used. The aim of this study was to evaluate the pain level in children under four months during vaccination according to pain management. METHODS: A six month observational study was performed in University Hospital of Amiens. A first part of the study focused on paediatricians of Picardie in order to assess their usual analgesic therapy during vaccination. A second part of the study evaluated pain level in children under four month during vaccination. Pain level was assessed with time of cry and acute pain in newborn infants scale (DAN, douleur aigüe du nouveau-né) according analgesic therapy (oral sucrose, local Lidocaïne-Prilocaïne), order of vaccines (the diphtheria, polio, and tetanus toxoids and acellular pertussis and Haemophilus influenzae type b (DPTaP-Hib) vaccine and the pneumococcal conjugate vaccine) and parents contact. RESULTS: 144 infants under four months receiving two vaccines with a DAN scale assessment were included in our study. No significant difference was observed between the different analgesic therapies. DAN score was lower for infants held by parents (DAN = 7) compared to infants in examination table (DAN = 8, p<0,05) during pneumococcal conjugate vaccine. DPTaP-Hib vaccine was less painful (Cry duration = 33 sec; DAN = 5,3) compared to pneumococcal conjugate vaccine (Cry duration = 61 sec; DAN = 7,1). At the end of visit, all the children included had a DAN value under 3 and did not need furthermore analgesic therapy. CONCLUSION: In this study, we don't observed an optimal analgesic therapy to decrease pain expression in infants during vaccination. But anticipation of pain procedure are essential and need an pain evaluation for each invasive care.AMIENS-BU Santé (800212102) / SudocSudocFranceF

Polyethylene bag wrapping to prevent hypothermia during percutaneous central venous catheter insertion in the preterm newborn under 32 weeks of gestation

International audienceAim: In preterm neonates, during nursing procedures, body temperature decreases. This study evaluates the interest of polyethylene bag wrapping to prevent this decrease during percutaneous central venous catheter (PCVC) insertion procedure, in preterm neonates under 32 weeks of gestation nursed in closed incubators. Methods: This prospective observational study compared two periods: [May 2009-September 2009]: ``without polyethylene bag wrapping'' and [October 2009-March 2010]: ``with polyethylene bag wrapping''. The main criterion was newborn skin temperature at the end of the procedure. Results: There was no difference between the two groups for skin temperature before the procedure (36.9 +/- 0.3 degrees C versus 36.9 +/- 0.3 degrees C; p = NS). The skin temperature at the end of the procedure was lower in the ``without bag wrapping'' group (36.0 +/- 0.5 degrees C) compared to the ``bag wrapping'' group (36.4 degrees C +/- 0.5 degrees C; p = 0.01). Furthermore, no skin temperature at the end was higher than 37.4 degrees C in the bag wrapping group. Conclusion: The use of a polyethylene bag was effective in decreasing skin temperature fall during a PCVC insertion procedure in our population. No side effects were observed. The benefit of prolonged wrapping or for shorter procedure should be evaluated

Estrogen receptor actions on vascular biology and inflammation: implications in vascular pathophysiology.

International audienceWhereas hormonal therapy (HT) may increase the risk of coronary heart disease (CHD) and stroke in menopausal women, epidemiological studies (protection in premenopausal women) suggest and experimental studies (prevention of fatty streak development in animals) demonstrate a major atheroprotective action of estradiol (E2). The understanding of the deleterious and beneficial effects of estrogens is thus required at both a cellular and molecular level. Both the endothelium and the immuno-inflammatory system play a key role in the development of fatty streak deposit as well as in the rupture of the atherosclerotic plaque. Whereas E2 favors an anti-inflammatory effect in vitro (cultured cells), it rather elicits a pro-inflammatory response in vivo at the level of several subpopulations of the immuno-inflammatory system, which could contribute to plaque destabilization. E2 promotes beneficial actions on the endothelium such as nitric oxide and prostacyclin production. E2 actions are essentially mediated by two molecular targets: estrogen receptor alpha (ER-alpha) and beta (ER-beta), but the former appears to mediate most of the actions of E2 on the endothelium and on the immune system. ER-alpha modulates target gene transcription through two activation functions (AF), AF-1 and AF-2, even though signalling via ER-alpha located at the plasma membrane (responsible for membrane-initiated steroid signalling (MISS)/(extra-genomic)) can also lead to an indirect effect on gene transcription. Recently, we demonstrated that ER-alpha AF-1 is not required for the vasculoprotective actions of E2, whereas it is necessary for the effects of E2 on its reproductive targets. These results suggest that selective estrogen receptor modulators stimulating ER-alpha with minimal activation of ER-alpha AF-1 could retain beneficial vascular actions, while minimizing the sexual effects