Roles of fibronectin isoforms in neonatal vascular development and matrix integrity

Fibronectin (FN) exists in two forms—plasma FN (pFN) and cellular FN (cFN). Although the role of FN in embryonic blood vessel development is well established, its function and the contribution of individual isoforms in early postnatal vascular development are poorly understood. Here, we employed a tamoxifen-dependent cFN inducible knockout (cFN iKO) mouse model to study the consequences of postnatal cFN deletion in smooth muscle cells (SMCs), the major cell type in the vascular wall. Deletion of cFN influences collagen deposition but does not affect life span. Unexpectedly, pFN translocated to the aortic wall in the cFN iKO and in control mice, possibly rescuing the loss of cFN. Postnatal pFN deletion did not show a histological aortic phenotype. Double knockout (dKO) mice lacking both, cFN in SMCs and pFN, resulted in postnatal lethality. These data demonstrate a safeguard role of pFN in vascular stability and the dispensability of the individual FN isoforms in postnatal vascular development. Complete absence of FNs in the dKOs resulted in a disorganized tunica media of the aortic wall. Matrix analysis revealed common and differential roles of the FN isoforms in guiding the assembly/deposition of elastogenic extracellular matrix (ECM) proteins in the aortic wall. In addition, we determined with two cell culture models that that the two FN isoforms acted similarly in supporting matrix formation with a greater contribution from cFN. Together, these data show that pFN exerts a critical role in safeguarding vascular organization and health, and that the two FN isoforms function in an overlapping as well as distinct manner to maintain postnatal vascular matrix integrity

A new model of development of the mammalian ovary and follicles

Ovarian follicular granulosa cells surround and nurture oocytes, and produce sex steroid hormones. It is believed that during development the ovarian surface epithelial cells penetrate into the ovary and develop into granulosa cells when associating with oogonia to form follicles. Using bovine fetal ovaries (n = 80) we identified a novel cell type, termed GREL for Gonadal Ridge Epithelial-Like. Using 26 markers for GREL and other cells and extracellular matrix we conducted immunohistochemistry and electron microscopy and chronologically tracked all somatic cell types during development. Before 70 days of gestation the gonadal ridge/ovarian primordium is formed by proliferation of GREL cells at the surface epithelium of the mesonephros. Primordial germ cells (PGCs) migrate into the ovarian primordium. After 70 days, stroma from the underlying mesonephros begins to penetrate the primordium, partitioning the developing ovary into irregularly-shaped ovigerous cords composed of GREL cells and PGCs/oogonia. Importantly we identified that the cords are always separated from the stroma by a basal lamina. Around 130 days of gestation the stroma expands laterally below the outermost layers of GREL cells forming a sub-epithelial basal lamina and establishing an epithelial-stromal interface. It is at this stage that a mature surface epithelium develops from the GREL cells on the surface of the ovary primordium. Expansion of the stroma continues to partition the ovigerous cords into smaller groups of cells eventually forming follicles containing an oogonium/oocyte surrounded by GREL cells, which become granulosa cells, all enclosed by a basal lamina. Thus in contrast to the prevailing theory, the ovarian surface epithelial cells do not penetrate into the ovary to form the granulosa cells of follicles, instead ovarian surface epithelial cells and granulosa cells have a common precursor, the GREL cell.Katja Hummitzsch, Helen F. Irving-Rodgers, Nicholas Hatzirodos, Wendy Bonner, Laetitia Sabatier, Dieter P. Reinhardt, Yoshikazu Sado, Yoshifumi Ninomiya, Dagmar Wilhelm and Raymond J. Rodger

Differential p97 adaptors and their role in cellular functions

The AAA ATPase p97/VCP functions in numerous cellular pathways. The molecular function of p97 in any of these pathways is specifically determined by the adaptor protein it is bound to. I am interested in studying new p97 functions, specifically their role related to cancer, such as apoptosis and cytoskeletal rearrangements. I initially focused on identifying the apoptotic adaptor of p97. I showed that PTEN is not the adaptor linking p97 to apoptosis through immunoprecipitation. Bioinformatics analysis revealed 14 potential p97 adaptors. FAF1 is the one involved in apoptosis, while Socius is a candidate for cytoskeletal rearrangements. I was able to outline a general strategy of how to determine and functionally classify candidate p97 interacting proteins

Fibrillins: functional aspects, assembly and expression patterns in development

Fibrillins constitute the major backbone of multi-functional microfibrils in elastic and non-elastic extracellular matrices. Proper expression, assembly and homeostasis mechanisms are central to the formation and function of these microfibrils. These properties are often compromised in pathological situations such as Marfan syndrome and other fibrillinopathies caused by mutations in fibrillins.While the developmental expression of fibrillin-1 and -2 has been intensely studied, fibrillin-3 is much less characterized in this regard. In this thesis, we analyze the developmental expression of human fibrillin-3 and compare it to that of other fibrillin isoforms. Fibrillin-3 is widely expressed in connective tissues of many organs as well as in close proximity to basement membranes of developing epithelia and endothelia. Generally, fibrillins are expressed in the same organs with a number of differences on the tissue level suggesting that fibrillin-3 fulfills specific functions in human development, both overlapping and distinct compared to the other fibrillin isoforms.In the present work, we identify fibronectin as a novel binding ligand for all fibrillins and demonstrate the requirement of a preassembled fibronectin network for fibrillin-1 network assembly and homeostasis. We show that multimerized C-terminal halves of all fibrillins and the N-terminal half of fibrillin-1 interact with high affinity with the collagen/gelatin binding region of fibronectin as well as with some lower affinity sites outside this region. In this study, we further characterize the role of heparin/heparan sulfate in microfibril assembly and its interaction with fibrillins. We demonstrate that fibrillin-2 and -3 interact with heparin/heparan sulfate and that fibrillin multimerization increases the avidity for heparin/heparan sulfate. We also show that heparin/heparan sulfate acts as a regulator of fibrillin homo- and heterotypic interactions, which are critical for microfibril assembly. Our results refine the sequence of events leading to microfibril assembly, consolidated in a new model.Fibrilline forme l'ossature des microfibrilles multifonctionnelles présentes dans les matrices élastiques et non-élastiques. Un bon fonctionnement des mécanismes d'expression, d'assemblé et d'homéostasie est essentiel à la formation de microfibrilles fonctionnelles. Ces mécanismes sont souvent altérés par des mutations dans les fibrillines lors de pathologies telles que le syndrome de Marfan et autres fibrillinopathies. Bien que l'expression développementale des fibrillines-1 et -2 eut été intensément étudiée, peu d'informations sur l'expression de fibrilline-3 sont connues. Dans cette thèse, nous analysons l'expression développementale de la protéine humaine fibrilline-3 et la comparons à l'expression des autres fibrillines. Fibrilline-3 est exprimée dans le tissue conjonctif de nombreux organes ainsi que proches des membranes basales épithéliales et endothéliales. Les trois fibrillines sont généralement exprimées dans les mêmes organes avec quelques différences au niveau tissulaire. Ces résultats suggèrent que fibrilline-3 ait des fonctions précises dans le développement humain, ces fonctions pouvant être identiques ou différentes des autres fibrillines.Dans cette étude, nous définissons fibronectine comme interagissant avec toutes les fibrillines et démontrons qu'un réseau préétabli de fibronectine est essentiel à la formation et l'homéostasie du réseau de fibrilline-1. Nous montrons que la moitié carboxylique multimérique de toutes les fibrillines ainsi que la moitié aminique de fibrilline-1 interagissent avec grande affinité avec la région d'interaction de collagène et gélatine de fibronectine ainsi qu'avec d'autres régions. Dans le travail présent, nous caractérisons aussi le rôle joué par héparine-sulfate d'héparane dans l'assemblé des microfibrilles ainsi que son intéraction avec les fibrillines. Nous démontrons que fibrilline-2 et -3 intéragissent avec héparine-sulfate d'héparane et que la multimerization des fibrillines augmente l'avidé pour l'héparine-sulfate d'héparane. Nous démontrons aussi que l'héparine-sulfate d'héparane agit en tant que régulateur des intéractions homo- et hétérotopiques entre fibrillines, ce qui est crucial pour l'assemblé des microfibrilles. La séquence d'évènements participant à la formation de microfibrilles est redéfinie par nos résultats et est présentée dans un nouveau modèle

Impact of drug storage systems: a quasi-experimental study with and without an automated-drug dispensing cabinet

International audienc

ROLE OF INDOLE-DERIVED UREMIC SOLUTES ON TISSUE FACTOR PRODUCTION VIA ARYL HYDROCARBON RECEPTOR PATHWAY

49th Congress of the European-Renal-Association/European-Dialysis-and-Transplant-Association (ERA-EDTA), Paris, FRANCE, MAY 24-27, 2012International audienceno abstrac

Elevation of endothelial microparticles in chronic renal failure: Role of uremic toxins

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Elevation of circulating endothelial microparticles in patients with chronic renal failure

International audienceBackground: Chronic renal failure patients are at high risk of cardiovascular events and display endothelial dysfunction, a critical element in the pathogenesis of atherosclerosis. Upon activation, the endothelium sheds microparticles, considered as markers of endothelial dysfunction that also behave as vectors of bioactive molecules. Aim: To measure plasma levels of endothelial microparticles (EMPs) in chronic renal failure patients (CRF), either undialyzed or hemodialyzed (HD), and to investigate the ability of uremic toxins to induce EMP release in vitro. Methods: Circulating EMPs were numerated by flow cytometry, after staining of platelet-free plasma with phycoerythrin (PE)-conjugated anti-CD144 (CD144+ EMP) or anti-CD146 (CD146+ EMP) monoclonal antibodies. Platelet MP (CD41+ PMP), leukocyte MP (CD45+ leukocyte microparticles (LMP)), and annexin-V+ MPs were also counted. In parallel, MPs were counted in supernatant of human umbilical vein endothelial cells incubated with uremic toxins [oxalate, indoxyl sulfate, p-cresol, and homocysteine (Hcy)], at concentrations found in patients. Results and conclusions: CD144+ EMP and CD146+ EMP levels were significantly higher in CRF and HD patients than in healthy subjects. Furthermore, annexin-V+ MPs were elevated in both groups of uremic patients, and CD41+ PMP and CD45+ LMP were increased in CRF and HD patients, respectively. In vitro, p-cresol and indoxyl sulfate significantly increased both CD146+ and annexin-V+ EMP release. Increased levels of circulating EMP in CRF and HD patients represent a new marker of endothelial dysfunction in uremia. The ability of p-cresol and indoxyl sulfate to increase EMP release in vitro suggests that specific uremic factors may be involved in EMP elevation in patients

A centralized automated-dispensing system in a French teaching hospital: return on investment and quality improvement

International audienceObjectives: To evaluate the return on investment (ROI) and quality improvement after implementation of a centralized automated-dispensing system after 8 years of use.Design: Prospective evaluation of ROI; before and after study to evaluate dispensing errors; user satisfaction questionnaire after 8 years of use.Setting: The study was conducted at a French teaching hospital in the pharmacy department, which is equipped with decentralized automated medication cabinets in the wards.Participants: Pharmacy staff (technicians and residents).Intervention(s): Implementation of a centralized automated-dispensing robot.Main outcome measure(s): The true ROI was prospectively and annually compared to estimated returns calculated after implementation and upgrade of the robot; dispensing errors determined by observation of global deliveries and the satisfaction of users based on a validated questionnaire were evaluated.Results: Following the upgrade, we found little difference for the ROI (+1.86%). The payback period increased by almost 3 years. There was a significant reduction of dispensing errors, from 2.9% to 1.7% (P < 0.001). User satisfaction of the robot by the pharmacy staff was reported (score of 5.52 ± 1.20 out of 7).Conclusions: These systems are worthwhile investments and largely contribute to improving the quality and safety of the medication process

BARD1 homozygous deletion, a possible alternative to BRCA1 mutation in basal breast cancer

International audienc