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Activation of the mineralocorticoid receptor (MR) in the heart is considered to be a cardiovascular risk factor. MR activation leads to heart hypertrophy and arrhythmia. In ventricular cardiomyocytes, aldosterone induces a profound remodeling of ion channel expression, in particular, an increase in the expression and activity of T-type voltage-gated calcium channels (T-channels). The molecular mechanisms immediately downstream from MR activation, which lead to the increased expression of T-channels and, consecutively, to an acceleration of spontaneous cell contractions in vitro, remain poorly investigated. Here, we investigated the putative role of a specific microRNA in linking MR activation to the regulation of T-channel expression and cardiomyocyte beating frequency. A screening assay identified microRNA 204 (miR-204) as one of the major upregulated microRNAs after aldosterone stimulation of isolated neonatal rat cardiomyocytes. Aldosterone significantly increased the level of miR-204, an effect blocked by the MR antagonist spironolactone. When miR-204 was overexpressed in isolated cardiomyocytes, their spontaneous beating frequency was significantly increased after 24 h, like upon aldosterone stimulation, and messenger RNAs coding T-channels (CaV3.1 and CaV3.2) were increased. Concomitantly, T-type calcium currents were significantly increased upon miR-204 overexpression. Specifically repressing the expression of miR-204 abolished the aldosterone-induced increase of CaV3.1 and CaV3.2 mRNAs, as well as T-type calcium currents. Finally, aldosterone and miR-204 overexpression were found to reduce REST-NRSF, a known transcriptional repressor of CaV3.2 T-type calcium channels. Our study thus strongly suggests that miR-204 expression stimulated by aldosterone promotes the expression of T-channels in isolated rat ventricular cardiomyocytes, and therefore, increases the frequency of the cell spontaneous contractions, presumably through the inhibition of REST-NRSF protein.

In vitro and animal studies point to autoantibodies against apolipoprotein A-1 (anti-apoA-1 IgG) as possible mediators of cardiovascular (CV) disease involving several mechanisms such as basal heart rate interference mediated by a mineralocorticoid receptor–dependent L-type calcium channel activation, and a direct pro-inflammatory effect through the engagement of the toll-like receptor (TLR) 2/CD14 complex. Nevertheless, the possible implication of these receptors in the pro-arrhythmogenic effect of anti-apoA-1 antibodies remains elusive. We aimed at determining whether CD14 and TLRs could mediate the anti-apoA-1 IgG chronotropic response in neonatal rat ventricular cardiomyocytes (NRVC). Blocking CD14 suppressed anti-apoA-1 IgG binding to NRVC and the related positive chronotropic response. Anti-apoA-1 IgG alone induced the formation of a TLR2/TLR4/CD14 complex, followed by the phosphorylation of Src, whereas aldosterone alone promoted the phosphorylation of Akt by phosphatidylinositol 3-kinase (PI3K), without affecting the chronotropic response. In the presence of both aldosterone and anti-apoA-1 IgG, the localization of TLR2/TLR4/CD14 was increased in membrane lipid rafts, followed by PI3K and Src activation, leading to an L-type calcium channel–dependent positive chronotropic response. Pharmacological inhibition of the Src pathway led to the decrease of L-type calcium channel activity and abrogated the NRVC chronotropic response. Activation of CD14 seems to be a key regulator of the mineralocorticoid receptor–dependent anti-apoA-1 IgG positive chronotropic effect on NRVCs, involving relocation of the CD14/TLR2/TLR4 complex into lipid rafts followed by PI3K and Src-dependent L-type calcium channel activation.

International audience

We previously reported that vascular endothelial growth factor induced vascular endothelial (VE)-cadherin tyrosine phosphorylation at Y685 in a Src-dependent manner in vitro. Here, we studied the occurrence of Y685 phosphorylation in vivo in the female reproductive tract because it is a unique model of physiological vascular remodeling dependent on vascular endothelial growth factor. We first developed and characterized an anti-phospho-specific antibody against the site Y685 of VE-cadherin to monitor VE-cadherin phosphorylation along the four phases of mouse estrous cycle, termed proestrus, estrus, metestrus, and diestrus. A dynamic profile of tyrosine phosphorylated proteins was observed in both uterus and ovary throughout mouse estrous cycle, including kinase Src, which was found highly active at the estrus phase. The extent of tyrosine phosphorylated VE-cadherin was low at proestrus but strongly increased at estrus and returned to baseline at metestrus and diestrus, suggesting a potent hormonal regulation of this specific process. Indeed, C57Bl/6 female mice treatment with pregnant mare serum gonadotropin and human chorionic gonadotropin confirmed a significant increase in phosphoY685-VE-cadherin compared with that in untreated mice. These results demonstrate that VE-cadherin tyrosine phosphorylation at Y685 is a physiological and hormonally regulated process in female reproductive organs. In addition, this process was concomitant with the early steps of vascular remodeling taking place at estrus stage, suggesting that phosphoY685-VE-cadherin is a biomarker of endothelial cell activation in vivo.

10. Ammar MR, Kassas N, Chasserot-Golaz S, et al. Lipids in regulated exocytosis: what are they doing? Front Endocrinol (Lausanne) 2013 ; 4 : 125. 4. Peters C, Bayer MJ, Buhler S, et al. Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion. Nature 2001 ; 409 : 581-8. 5. Strasser B, Iwaszkiewicz J, Michielin O, Mayer A. The V-ATPase proteolipid cylinder promotes the lipidmixing stage of SNARE-dependent fusion of yeast vacuoles. EMBO J 2011 ; 30 : 4126-41. 6. Hiesinger PR, Fayyazuddin A, Mehta SQ, et al. The v-ATPase V(0) subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila. Cell 2005 ; 121 : 607-20. 7. Liegeois S, Benedetto A, Garnier JM, et al. The V0-ATPase mediates apical secretion of exosomes REFERENCES

Endothelial dysfunction is a hallmark for vascular diseases. It is often seen in patients with coronary artery disease, diabetes, hypertension, rheumatoid arthritis, systemic vasculitis, and cancer. Identification of serologic markers that are associated with disease activity to diagnose or predict relapse is a challenging task. At the cellular level, endothelial dysfunction is the result of activation of several signaling pathways due to increased levels of cytokines associated with the disease. Endothelium integrity is dependent upon the adhesive function of the major molecule located at endothelial adherens junctions called vascular endothelial (VE)-cadherin. As in the case of other members of the cadherin family, VE-cadherin is able to mediate homotypic types of endothelial cellular interactions in a Ca2+-dependent manner and to link the underlying cytoskeleton. In conditions mimicking endothelial cell activation, the junctional complexes are subjected to posttranslational modifications such as phosphorylations or proteolysis reactions mediated by kinases and proteases, respectively, which are supposed to weaken the junctional strength. This chapter summarizes recent studies on VE-cadherin structural modifications in endothelial biology having potential applications in disease management and patient care.

Context: The circadian clock represents the body's molecular time-keeping system. Recent findings revealed strong changes of clock gene expression in various types of human cancers. Objective: Due to emerging evidence on the connection between the circadian oscillator, cell cycle, and oncogenic transformation, we aimed to characterize the circadian clockwork in human benign and malignant thyroid nodules. Design: Clock transcript levels were assessed by quantitative RT-PCR in thyroid tissues. To provide molecular characteristics of human thyroid clockwork, primary thyrocytes established from normal or nodular thyroid tissue biopsies were subjected to in vitro synchronization with subsequent clock gene expression analysis by circadian bioluminescence reporter assay and by quantitative RT-PCR. Results: The expression levels of the Bmal1 were up-regulated in tissue samples of follicular thyroid carcinoma (FTC), and in papillary thyroid carcinoma (PTC), as compared with normal thyroid and benign nodules, whereas Cry2 was down-regulated in FTC and PTC. Human thyrocytes derived from normal thyroid tissue exhibited high-amplitude circadian oscillations of Bmal1-luciferase reporter expression and endogenous clock transcripts. Thyrocytes established from FTC and PTC exhibited clock transcript oscillations similar to those of normal thyroid tissue and benign nodules (except for Per2 altered in PTC), whereas cells derived from poorly differentiated thyroid carcinoma exhibited altered circadian oscillations. Conclusions: This is the first study demonstrating a molecular makeup of the human thyroid circadian clock. Characterization of the thyroid clock machinery alterations upon thyroid nodule malignant transformation contributes to understanding the connections between circadian clocks and oncogenic transformation. Moreover, it might help in improving the thyroid nodule preoperative diagnostics.

International audience; To identify patients with autoimmune diseases who are at high risk of developing vascular cell dysfunction, early biomarkers must be identified. This study was designed to detect and characterize circulating autoantibodies to VE-cadherin (AAVEs) in patients with early-stage autoimmune diseases. An enzyme-linked immunosorbent assay (ELISA) was developed to capture autoantibodies, using a recombinant human VE-cadherin fragment covering the extracellular domains as a target antigen. AAVEs specificity for the target antigen was confirmed by western blotting. Basal AAVEs levels were determined for healthy donors (n=75). Sera from patients (n=100) with various autoimmune diseases, including rheumatoid arthritis (n=23), systemic lupus erythematosus (SLE, n=31), systemic sclerosis (n=30), and Behçet's disease (BD, n=16) were also tested. Levels of AAVEs were significantly higher in rheumatoid arthritis (P

Vessel abnormalities are among the most important features in malignant glioma. Vascular endothelial (VE)-cadherin is of major importance for vascular integrity. Upon cytokine challenge, VE-cadherin structural modifications have been described including tyrosine phosphorylation and cleavage. The goal of this study was to examine whether these events occurred in human glioma vessels. We demonstrated that VE-cadherin is highly expressed in human glioma tissue and tyrosine phosphorylated at site Y(685), a site previously found phosphorylated upon VEGF challenge, via Src activation. In vitro experiments showed that VEGF-induced VE-cadherin phosphorylation, preceded the cleavage of its extracellular adhesive domain (sVE, 90 kDa). Interestingly, metalloproteases (MMPs) secreted by glioma cell lines were responsible for sVE release. Because VEGF and MMPs are important components of tumor microenvironment, we hypothesized that VE-cadherin proteolysis might occur in human brain tumors. Analysis of glioma patient sera prior treatment confirmed the presence of sVE in bloodstream. Furthermore, sVE levels studied in a cohort of 53 glioma patients were significantly predictive of the overall survival at three years (HR 0.13 [0.04; 0.40] p ≤ 0.001), irrespective to histopathological grade of tumors. Altogether, these results suggest that VE-cadherin structural modifications should be examined as candidate biomarkers of tumor vessel abnormalities, with promising applications in oncology.

Corticosteroids have been involved in the genesis of ventricular arrhythmias associated with pathological heart hypertrophy, although molecular mechanisms responsible for these effects have not been completely explained. Because mineralocorticoid receptor (MR) antagonists have been demonstrated to be beneficial on the cardiac function, much attention has been given to the action of aldosterone on the heart. However, we have previously shown that both aldosterone and corticosterone in vitro induce a marked acceleration of the spontaneous contractions, as well as a significant cell hypertrophy in isolated neonate rat ventricular cardiomyocytes. Moreover, a beneficial role of the steroid hormone dehydroepiandrosterone (DHEA) has been also proposed, but the mechanism of its putative cardioprotective function is not known. We found that DHEA reduces both the chronotropic and the hypertrophic responses of cardiomyocytes upon stimulation of MR and glucocorticoid receptor (GR) in vitro. DHEA inhibitory effects were accompanied by a decrease of T-type calcium channel expression and activity, as assessed by quantitative PCR and the patch-clamp technique. Prevention of cell hypertrophy by DHEA was also revealed by measuring the expression of A-type natriuretic peptide and BNP. The kinetics of the negative chronotropic effect of DHEA, and its sensitivity to actinomycin D, pointed out the presence of both genomic and nongenomic mechanisms of action. Although the genomic action of DHEA was effective mostly upon MR activation, its rapid, nongenomic response appeared related to DHEA antioxidant properties. On the whole, these results suggest new mechanisms for a putative cardioprotective role of DHEA in corticosteroid-associated heart diseases.

Objective Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that principally attacks synovial joints. However, accelerated atherosclerosis and increased cardiovascular morbidity and mortality are major clinical consequences of endothelial dysfunction in RA patients. Tumor necrosis factor α (TNFα) is the major mediator of inflammation in RA, related to vascular injury by targeting VE-cadherin, an endothelium-specific adhesion molecule of vital importance for endothelium integrity and angiogenesis. We undertook this study to examine the mechanisms regulating VE-cadherin processing by TNFα and their occurrence in RA. Methods Human umbilical vein endothelial cells were used in primary culture and treated with recombinant TNFα to study VE-cadherin cleavage. Cell lysates and conditioned media were analyzed by Western blotting for VE-cadherin cytoplasmic domain and extracellular domain (VE-90) generation, respectively. VE-90 was analyzed at baseline and at the 1-year followup in sera from 63 RA patients (from the Very Early Rheumatoid Arthritis cohort) with disease duration of

INTRODUCTION Autoimmune mediated inflammation may play a role in atherosclerosis and atherothrombosis essential features for cardiovascular disease (CVD) manifestations. Anti Apolipoprotein A 1 (ApoA 1) IgG autoantibodies are an independent predictor of poor cardiovascular outcome in different clinical settings promote inflammation and atherogenesis and are associated with increased atherosclerotic plaque vulnerability in humans and mice. AIM To determine whether anti ApoA 1 IgG are associated with Tissue Factor (TF) expression a key regulator of the extrinsic coagulation pathway involved in atherothrombosis responsible for the majority of acute CVD manifestations. METHODS Atherothrombosis features were explored in vivo by immunohistochemical TF staining on human carotid atherosclerotic plaques from (n = 102) patients with severe carotid stenosis and in vitro on human monocyte derived macrophages (HMDM) TF expression and pro coagulant activity detected by cytofluorimetry and chromogenic assay respectively. RESULTS In vivo atherosclerotic biopsies derived from patients with high circulating levels of anti apoA 1 IgG (n = 20) displayed a higher TF expression when compared to biopsies from patients with low anti apoA 1 IgG levels (9.3 vs 3.3 p

Rationnel La majorite des organismes vivants possedent des oscillateurs circadiens. Ces horloges sont presentes dans la majorite des cellules du corps. Les oscillateurs circadiens sont fonctionnels dans les cellules en culture primaire ou immortalisees. Une evidence croissante, apportee par des etudes recentes, etablit l'existence d'une connexion entre le nombre de syndromes metaboliques, comme l'obesite et le diabete, et l'horloge circadienne. Notre but principal etait d'apporter de nouveaux elements pour mieux comprendre comment le systeme circadien agit sur la fonction du pancreas endocrine chez l'Homme. Dans cette etude, nous cherchons a explorer l'impact de l'oscillateur pancreatique sur la fonction des cellules de l'ilot. Materiels et methodes Nous avons etabli un modele de visualisation de l'oscillation circadienne de l'ilot humain en enregistrant la bioluminescence in vivo, a long terme, dans des ilots pancreatiques entiers ou dans les cellules primaires de l'ilot. Resultats Les ilots humains intacts ainsi que les cellules d'ilots disperses synchronises in vitro exhibent des oscillations circadiennes du reporteur Bmal1-luciferase avec une longueur de periode de respectivement 23.6 et 23.9 heures. Les transcrits endogenes de BMAL1 and CRY1 ont le profile circadien pendant 48 heures, qui sont en antiphase aux seines de REV-ERBα, PER1, PER2, PER3 et DBP. HNF1A et PDX1 exhibent des oscillations circadiennes faibles avec une phase correspondant a celle du REV-ERBα. Les cellules d'ilots disperses montrent egalement des oscillations fortes au niveau de la population ainsi qu'au niveau des cellules isolees. Les horloges des cellules beta et non-beta fonctionnent de maniere synchronisee. Conclusion Nous avons demontre et caracterise, pour la premiere fois, la presence des oscillateurs circadiens autonomes dans les ilots pancreatiques humains. La dissection de la fonction de l'oscillateur pancreatique physiologique ou diabetique pourrait avancer la comprehension du mecanisme liant le systeme circadien pancreatique, les desordres d'origines metaboliques et le diabete de type 2.

BACKGROUND: Vascular endothelial (VE)-cadherin is an endothelial specific adhesion molecule of vital importance for the maintenance of endothelium integrity. Because gliomas are hypervascular tumors, the present study was designed to address the question whether Vascular endothelial-cadherin (VE-cadherin), a protein exclusively expressed in endothelial cells, could be a potential candidate biomarker for clinical use in the setting of gliomas. METHODS: Fifty-six patients newly diagnosed glioma who underwent radiochemotherapy were enrolled in this retrospective study. Serum samples were collected prior treatment. Therapeutic response to Temodal was observed by RMI in 24 patients (25 oligodendroglioma, 2 glioblastoma multiform) median age 42.15 (range 23-65) while 32 patients were resistant (5 anaplastic oligodendroglioma, 9 anaplastic astrocytomas and 17 glioblastomas multiforme), median age 51.9 (range 19-84). sVE-cadherin levels was evaluated by westernblotting and sandwich ELISA. Data were analyzed using logistic regression and receiver operating characteristics (ROC) curve analysis. RESULTS: VE-cadherin 90 kDa extracellular domain was detected in human serum by western blot and its accurate detection was dependent upon a pre-dilution in a detergent-containing buffer (Triton X-100). Of importance, we found that sVE-cadherin was carried by human lipoproteins and that the detergent allowed to separate both entities to increase protein detectability. Interestingly, we found that sVE cadherin was significantly lower in the non responders group as compared to responders. We developped an ELISA that clearly discriminate patients NR (0.669+/-0.104 AU) from patients R (0.975+/-0.098 AU (p=0.0024). Receiver-operating characteristic (ROC) curve analysis revealed that the predictive cut-off value of serum VE-cadherin for chemoresistance was 0.735 AU (area-under-the-curve 0.82; 95% confidence interval 0.71-0.87; maximal accuracy 0.875) Patients lipid profile (total cholesterol, triglycerides, LDL and HDL) were not significantly different between the two groups and could not account for the differences observed in serum VE-cadherin levels. CONCLUSIONS: We show for the first time that the lipoprotein component of serum — which is most often ignored in protein diagnostics — contains proteins that can unexpectedly serve as biomarkers for disease. This finding may open completely new avenues of biomarker research. Second, we demonstrate, one candidate marker (soluble VE-cadherin) to follow glioma therapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2724.

Aims/hypothesis Following on from the emerging importance of the pancreas circadian clock on islet function and the development of type 2 diabetes in rodent models, we aimed to examine circadian gene expression in human islets. The oscillator properties were assessed in intact islets as well as in beta cells. Methods We established a system for long-term bioluminescence recording in cultured human islets, employing lentivector gene delivery of the core clock gene Bmal1 (also known as Arntl)-luciferase reporter. Beta cells were stably labelled using a rat insulin2 promoter fluorescent construct. Single-islet/cell oscillation profiles were measured by combined bioluminescence–fluorescence time-lapse microscopy. Results Human islets synchronised in vitro exhibited self-sustained circadian oscillations of Bmal1-luciferase expression at both the population and single-islet levels, with period lengths of 23.6 and 23.9 h, respectively. Endogenous BMAL1 and CRY1 transcript expression was circadian in synchronised islets over 48 h, and antiphasic to REV-ERBα (also known as NR1D1), PER1, PER2, PER3 and DBP transcript circadian profiles. HNF1A and PDX1 exhibited weak circadian oscillations, in phase with the REV-ERBα transcript. Dispersed islet cells were strongly oscillating as well, at population and single-cell levels. Importantly, beta and non-beta cells revealed oscillatory profiles that were well synchronised with each other. Conclusions/interpretation We provide for the first time compelling evidence for high-amplitude cell-autonomous circadian oscillators displayed in human pancreatic islets and in dispersed human islet cells. Moreover, these clocks are synchronised between beta and non-beta cells in primary human islet cell cultures. Electronic supplementary material The online version of this article (doi:10.1007/s00125-012-2779-7) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

L’endothelium vasculaire est la premiere couche de cellules en contact avec le flux sanguin. Au cours de processus pathologiques, les cellules endotheliales subissent des remaniements notoires dus a la presence de nombreux facteurs de croissance. Dans ce cas, l’integrite de l’endothelium, assuree principalement par les jonctions adherentes endotheliales dont la V(ascular) E(ndothelial)-cadherine est la proteine cle, est perturbe. Notre objectif est de mieux comprendre les modifications des jonctions endotheliales et plus particulierement de la VEcadherine. La phospho-VE-cadherine est une caracteristique des cellules endotheliales activees in vitro. Notre laboratoire a montre l’existence d’une forme phosphorylee de VE-cadherine in vivo au cours de l’angiogenese physiologique (stimulation hormonale). Des tyrosines importantes ont ete identifiees : la tyrosine 685, qui est la cible directe de la tyrosine kinase Src en reponse au VEGF ainsi que les tyrosines 658 et 731. De plus, la VE-cadherine peut etre sensible a la proteolyse. Dans ce travail, la phosphorylation de la VE-cadherine a ete etudiee dans un contexte pathologique d’inflammation et de cancer. Pour cela, deux modeles murins de tumeurs hepatiques ou cerebrales ont ete analyses. Par l’utilisation d’anticorps antiphosphosite specifiques, j’ai demontre l’existence in vivo d’une forme phosphorylee de VE-cadherine sur le site Y658. L’analyse de l’inhibition d’une tyrosine kinase particuliere, SYK, par si RNA ainsi que par des experiences de phosphorylation de la VE-cadherine in vitro suggerent fortement l’implication de SYK dans cette phosphorylation. En parallele, dans le cadre d’une etude sur la polyarthite rhumatoide, nous avons mis en evidence la presence d’une forme circulante de VE-cadherine correspondant a son domaine extracellulaire clive. Cette etude a pour but d’evaluer si la VE-cadherine circulante peut etre un marqueur d’une inflammation a distance. Les resultats sur 75 patients, en cours d’analyse statistique, seront exposes dans cette presentation. La VE-cadherine, tant par sa phosphorylation que par sa presence dans les fluides biologiques, pourrait etre une signature de l’endothelium active (phosphorylation) et/ou agresse (proteolyse).