Publications by authors named "Jean Ripoche"

31 Publications

In vitro comparison of three percutaneous atrial septal defect closure devices for endothelialisation and haemocompatibility.

Arch Cardiovasc Dis 2020 Aug - Sep;113(8-9):503-512. Epub 2020 Jul 24.

Université de Bordeaux, 33000 Bordeaux, France; Bioingénierie Tissulaire, U1026, INSERM, 33000 Bordeaux, France; CHU de Bordeaux, 33000 Bordeaux, France; CIC 1401, CHU de Bordeaux, 33000 Bordeaux, France.

Background: Percutaneous device closure of atrial septal defect (ASD) is the gold-standard treatment, but several delayed complications may occur as a result of incomplete device endothelialisation.

Aims: In this in vitro study, we compared three ASD closure devices [Nit-Occlud® ASD-R (device 1); Hyperion™ ASDO (device 2); and Amplatzer™ Septal Occluder (device 3)] in terms of the endothelialisation process, using human endothelial progenitors cells (EPCs), and haemocompatibility.

Methods: EPCs from umbilical cord blood were extracted, cultured and characterised. Device samples were seeded with 100,000 cells/cm. EPC adhesion was investigated at 3 and 24hours, and EPC proliferation was monitored, which allowed longitudinal follow-up (days 1-12). Haemocompatibility of device samples was assessed using a complement C3a assay and platelet and coagulation activation.

Results: With regard to EPC adhesion and proliferation, no statistically significant differences were found between the three devices. We observed for each device a significant time-dependent EPC proliferation, appearing at day 8 for devices 2 and 3 and day 10 for device 1. No complement or platelet activation occurred within 15minutes of contact with devices. However, there was minimal activation of coagulation for the three devices.

Conclusions: In this in vitro study we showed that, despite the three ASD occluders having different device designs and coatings, adhesion and proliferation of human endothelial cells was similar for all devices. This should be further confirmed by similar studies including shear stress forces and anti-thrombotic treatments.
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http://dx.doi.org/10.1016/j.acvd.2020.03.022DOI Listing
September 2020

CD154 Induces Interleukin-6 Secretion by Kidney Tubular Epithelial Cells under Hypoxic Conditions: Inhibition by Chloroquine.

Mediators Inflamm 2020 31;2020:6357046. Epub 2020 Jan 31.

INSERM, UMR1026 Bioingénierie Tissulaire (Biotis), Université de Bordeaux, F-33000 Bordeaux, France.

Inflammation is a major contributor to tubular epithelium injury in kidney disorders, and the involvement of blood platelets in driving inflammation is increasingly stressed. CD154, the ligand of CD40, is one of the mediators supporting platelet proinflammatory properties. Although hypoxia is an essential constituent of the inflammatory reaction, if and how platelets and CD154 regulate inflammation in hypoxic conditions remain unclear. Here, we studied the control by CD154 of the proinflammatory cytokine interleukin- (IL-) 6 secretion in short-term oxygen (O) deprivation conditions, using the HK-2 cell line as a kidney tubular epithelial cell (TEC) model. IL-6 secretion was markedly stimulated by CD154 after 1 to 3 hours of hypoxic stress. Both intracellular IL-6 expression and secretion were stimulated by CD154 and associated with a strong upregulation of IL-6 mRNA and increased transcription. Searching for inhibitors of CD154-mediated IL-6 production by HK-2 cells in hypoxic conditions, we observed that chloroquine, a drug that has been repurposed as an anti-inflammatory agent, alleviated this induction. Therefore, CD154 is a potent early stimulus for IL-6 secretion by TECs in O deprivation conditions, a mechanism likely to take part in the deleterious inflammatory consequences of platelet activation in kidney tubular injury. The inhibition of CD154-induced IL-6 production by chloroquine suggests the potential usefulness of this drug as a therapeutic adjunct in conditions associated with acute kidney injury.
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http://dx.doi.org/10.1155/2020/6357046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7013356PMC
November 2020

New factors for protein transport identified by a genome-wide CRISPRi screen in mammalian cells.

J Cell Biol 2019 11 5;218(11):3861-3879. Epub 2019 Sep 5.

University of Cambridge Metabolic Research Laboratories, Wellcome Trust-Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge, UK

Protein and membrane trafficking pathways are critical for cell and tissue homeostasis. Traditional genetic and biochemical approaches have shed light on basic principles underlying these processes. However, the list of factors required for secretory pathway function remains incomplete, and mechanisms involved in their adaptation poorly understood. Here, we present a powerful strategy based on a pooled genome-wide CRISPRi screen that allowed the identification of new factors involved in protein transport. Two newly identified factors, TTC17 and CCDC157, localized along the secretory pathway and were found to interact with resident proteins of ER-Golgi membranes. In addition, we uncovered that upon TTC17 knockdown, the polarized organization of Golgi cisternae was altered, creating glycosylation defects, and that CCDC157 is an important factor for the fusion of transport carriers to Golgi membranes. In conclusion, our work identified and characterized new actors in the mechanisms of protein transport and secretion and opens stimulating perspectives for the use of our platform in physiological and pathological contexts.
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http://dx.doi.org/10.1083/jcb.201902028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829651PMC
November 2019

Correction to: Blood platelets and sepsis pathophysiology: A new therapeutic prospect in critically ill patients?

Ann Intensive Care 2018 02 28;8(1):32. Epub 2018 Feb 28.

INSERM U1026, BioTis, Univ. Bordeaux, 33000, Bordeaux, France.

Upon publication of the original article [1], it was noticed that the title was incorrect. Instead of 'critical', it should read 'critically', and therefore, the correct title should be.
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http://dx.doi.org/10.1186/s13613-018-0378-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5833331PMC
February 2018

Unconventional secretion of FABP4 by endosomes and secretory lysosomes.

J Cell Biol 2018 02 6;217(2):649-665. Epub 2017 Dec 6.

Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA

An appreciation of the functional properties of the cytoplasmic fatty acid binding protein 4 (FABP4) has advanced with the recent demonstration that an extracellular form secreted by adipocytes regulates a wide range of physiological functions. Little, however, is known about the mechanisms that mediate the unconventional secretion of FABP4. Here, we demonstrate that FABP4 secretion is mediated by a membrane-bounded compartment, independent of the conventional endoplasmic reticulum-Golgi secretory pathway. We show that FABP4 secretion is also independent of GRASP proteins, autophagy, and multivesicular bodies but involves enclosure within endosomes and secretory lysosomes. We highlight the physiological significance of this pathway with the demonstration that an increase in plasma levels of FABP4 is inhibited by chloroquine treatment of mice. These findings chart the pathway of FABP4 secretion and provide a potential therapeutic means to control metabolic disorders associated with its dysregulated secretion.
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http://dx.doi.org/10.1083/jcb.201705047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800802PMC
February 2018

Blood platelets and sepsis pathophysiology: A new therapeutic prospect in critically [corrected] ill patients?

Ann Intensive Care 2017 12 1;7(1):115. Epub 2017 Dec 1.

INSERM U1026, BioTis, Univ. Bordeaux, 33000, Bordeaux, France.

Beyond haemostasis, platelets have emerged as versatile effectors of the immune response. The contribution of platelets in inflammation, tissue integrity and defence against infections has considerably widened the spectrum of their role in health and disease. Here, we propose a narrative review that first describes these new platelet attributes. We then examine their relevance to microcirculatory alterations in multi-organ dysfunction, a major sepsis complication. Rapid progresses that are made on the knowledge of novel platelet functions should improve the understanding of thrombocytopenia, a common condition and a predictor of adverse outcome in sepsis, and may provide potential avenues for management and therapy.
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http://dx.doi.org/10.1186/s13613-017-0337-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709271PMC
December 2017

A Role for CD154, the CD40 Ligand, in Granulomatous Inflammation.

Mediators Inflamm 2017 12;2017:2982879. Epub 2017 Jul 12.

INSERM U1026, Université de Bordeaux, 33000 Bordeaux, France.

Granulomatous inflammation is a distinctive form of chronic inflammation in which predominant cells include macrophages, epithelioid cells, and multinucleated giant cells. Mechanisms regulating granulomatous inflammation remain ill-understood. CD154, the ligand of CD40, is a key mediator of inflammation. CD154 confers a proinflammatory phenotype to macrophages and controls several macrophagic functions. Here, we studied the contribution of CD154 in a mouse model of toxic liver injury with carbon tetrachloride and a model of absorbable suture graft. In both models, granulomas are triggered in response to endogenous persistent liver calcified necrotic lesions or by grafted sutures. CD154-deficient mice showed delayed clearance of carbon tetrachloride-induced liver calcified necrotic lesions and impaired progression of suture-induced granuloma. In vitro, CD154 stimulated phagocytosis of opsonized erythrocytes by macrophages, suggesting a potential mechanism for the altered granulomatous inflammation in CD154KO mice. These results suggest that CD154 may contribute to the natural history of granulomatous inflammation.
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http://dx.doi.org/10.1155/2017/2982879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5529663PMC
May 2018

CD40 signaling and hepatic steatosis: Unanticipated links.

Clin Res Hepatol Gastroenterol 2017 Sep 15;41(4):357-369. Epub 2016 Dec 15.

INSERM U1026, Université de Bordeaux, 33000 Bordeaux, France. Electronic address:

Obesity predisposes to an increased risk of nonalcoholic fatty liver disease (NAFLD). Hepatic steatosis is the key pathological feature of NAFLD and has emerged as a metabolic disorder in which innate and adaptive arms of the immune response play a central role in disease pathogenesis. Recent studies have revealed unexpected relationships between CD40 signaling and hepatic steatosis in high fat diet rodent models. CD154, the ligand of CD40, is a mediator of inflammation and controls several critical events of innate and adaptive immune responses. In the light of these reports, we discuss potential links between CD40 signaling and hepatic steatosis in NAFLD.
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http://dx.doi.org/10.1016/j.clinre.2016.11.002DOI Listing
September 2017

CD154 Induces Matrix Metalloproteinase-9 Secretion in Human Podocytes.

J Cell Biochem 2016 12 5;117(12):2737-2747. Epub 2016 May 5.

INSERM U1026, Université de Bordeaux, F-33076 Bordeaux, France.

Matrix remodeling is a key feature of glomerulosclerosis secondary to diabetes or hypertension. Podocytes contribute to glomerular basement membrane (GBM) turnover by producing matrix components and matrix remodelling enzymes, including matrix metalloproteinases (MMPs). The CD40/CD154 signaling pathway modulates matrix remodeling through the synthesis of MMPs and tissue inhibitors of MMPs. Platelets are a primary blood reservoir of CD154. Here we studied, the impact of the CD154/CD40 pathway on MMP-9 expression by cultured human podocytes. The role of CD40/CD154 was evaluated upon exposure of podocytes to recombinant human CD154 (rhCD154) or activated platelet supernatants from healthy human subjects. We first showed by protein and mRNA expression that CD40 was synthesized by podocytes and detectable on kidney tissue sections. CD40 expression was acquired during podocyte differentiation and enhanced upon exposure to rhCD154. In podocytes, rhCD154 induced an increase of MMP-9 production as shown by RT-PCR, Western blot and and gelatin zymography. Activated platelet supernatants induced MMP-9 mRNA synthesis in podocytes, an effect reduced by anti-CD40 antibody. Our results underscore a potential role for platelets through the CD40/CD154 signaling pathway in the control of GBM synthesis and degradation, via its regulatory role on MMP-9 production. CD154 secretion by activated platelets may contribute to GBM alterations in proteinuric nephropathies. J. Cell. Biochem. 117: 2737-2747, 2016. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jcb.25571DOI Listing
December 2016

Beneficial Effect of Covalently Grafted α-MSH on Endothelial Release of Inflammatory Mediators for Applications in Implantable Devices.

PLoS One 2016 3;11(3):e0150706. Epub 2016 Mar 3.

Univ. Bordeaux, CBMN, UMR 5248, F-33600, Pessac, France.

Intravascular devices for continuous glucose monitoring are promising tools for the follow up and treatment of diabetic patients. Limiting the inflammatory response to the implanted devices in order to achieve better biocompatibility is a critical challenge. Herein we report on the production and the characterization of gold surfaces covalently derivatized with the peptide α-alpha-melanocyte stimulating hormone (α-MSH), with a quantifiable surface density. In vitro study demonstrated that the tethered α-MSH is able to decrease the expression of an inflammatory cytokine produced by endothelial cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150706PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4777356PMC
August 2016

Kinetic eGFR and Novel AKI Biomarkers to Predict Renal Recovery.

Clin J Am Soc Nephrol 2015 Nov 4;10(11):1900-10. Epub 2015 Sep 4.

Departments of Anesthesia and Intensive Care II, INSERM U1034, Cardiovascular Adaptation to Ischemia, University of Bordeaux, Pessac, France.

Background And Objectives: Prompt recognition of severe renal impairment could improve the early management of critically ill patients. We compared the value of kinetic eGFR, plasma neutrophil gelatinase-associated lipocalin (NGAL), and urine tissue inhibitor of metalloproteinase-2 and urine insulin-like growth factor-binding protein 7 ([TIMP-2]*[IGFBP7]) in predicting short-term recovery from AKI and major adverse kidney events.

Design, Setting, Participants, & Measurements: During the 6-month study period, 245 patients were admitted to our intensive care unit. This study included 57 consecutive patients presenting with AKI within the first 24 hours after admission. AKI markers were evaluated at inclusion (day 0) and 24 hours later (day 1). Kinetic eGFR was calculated on day 1 according to serum creatinine evolution. Renal recovery was defined as normalization of serum creatinine with reversal of oliguria within 48 hours. Major adverse kidney events included death, need for RRT, or persistence of renal dysfunction at hospital discharge.

Results: Plasma NGAL and [TIMP-2]*[IGFBP7] predicted renal recovery, with area under the receiver-operating characteristic curve (AUC-ROC) values between 0.70 and 0.79 at inclusion. Although plasma NGAL values frequently reached the maximal measurement range, their decrease on day 1 predicted recovery. The kinetic eGFR calculation after initial resuscitation provided the best AUC-ROC value for renal recovery, at 0.87. The best predictions for major adverse kidney events were provided by [TIMP-2]*[IGFBP7] and kinetic eGFR (equal AUC-ROCs of 0.81). Combining AKI markers in addition to clinical prediction models improved the discrimination and reclassification of patients who will recover from AKI or suffer from major adverse kidney events.

Conclusions: Biomarkers of kidney damage predicted short-term renal recovery and major adverse kidney events for an unselected cohort of critically ill patients. Calculating the kinetic eGFR imposed a delay after initial resuscitation but provided a good diagnostic and prognostic approach. The utility of functional and damage AKI marker combinations in addition to clinical information requires validation in larger prospective studies.
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http://dx.doi.org/10.2215/CJN.12651214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4633802PMC
November 2015

New frontiers for platelet CD154.

Exp Hematol Oncol 2015 1;4. Epub 2015 Mar 1.

INSERM U1026, and Université de Bordeaux, F-33000 Bordeaux, France.

The role of platelets extends beyond hemostasis. The pivotal role of platelets in inflammation has shed new light on the natural history of conditions associated with acute or chronic inflammation. Beyond the preservation of vascular integrity, platelets are essential to tissue homeostasis and platelet-derived products are already used in the clinics. Unanticipated was the role of platelets in the adaptative immune response, allowing a renewed conceptual approach of auto-immune diseases. Platelets are also important players in cancer growth and dissemination. Platelets fulfill most of their functions through the expression of still incompletely characterized membrane-bound or soluble mediators. Among them, CD154 holds a peculiar position, as platelets represent a major source of CD154 and as CD154 contributes to most of these new platelet attributes. Here, we provide an overview of some of the new frontiers that the study of platelet CD154 is opening, in inflammation, tissue homeostasis, immune response, hematopoiesis and cancer.
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http://dx.doi.org/10.1186/s40164-015-0001-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4355125PMC
March 2015

IQ domain GTPase-activating protein 1 is involved in shear stress-induced progenitor-derived endothelial cell alignment.

PLoS One 2013 22;8(11):e79919. Epub 2013 Nov 22.

Bioingénierie Tissulaire, Université de Bordeaux, U 1026, F-33000 Bordeaux, France ; Bioingénierie Tissulaire, U1026, INSERM, Bordeaux, France.

Shear stress is one of mechanical constraints which are exerted by blood flow on endothelial cells (ECs). To adapt to shear stress, ECs align in the direction of flow through adherens junction (AJ) remodeling. However, mechanisms regulating ECs alignment under shear stress are poorly understood. The scaffold protein IQ domain GTPase activating protein 1 (IQGAP1) is a scaffold protein which couples cell signaling to the actin and microtubule cytoskeletons and is involved in cell migration and adhesion. IQGAP1 also plays a role in AJ organization in epithelial cells. In this study, we investigated the potential IQGAP1 involvement in the endothelial cells alignment under shear stress. Progenitor-derived endothelial cells (PDECs), transfected (or not) with IQGAP1 small interfering RNA, were exposed to a laminar shear stress (1.2 N/m(2)) and AJ proteins (VE-cadherin and β-catenin) and IQGAP1 were labeled by immunofluorescence. We show that IQGAP1 is essential for ECs alignment under shear stress. We studied the role of IQGAP1 in AJs remodeling of PDECs exposed to shear stress by studying cell localization and IQGAP1 interactions with VE-cadherin and β-catenin by immunofluorescence and Proximity Ligation Assays. In static conditions, IQGAP1 interacts with VE-cadherin but not with β-catenin at the cell membrane. Under shear stress, IQGAP1 lost its interaction from VE-cadherin to β-catenin. This "switch" was concomitant with the loss of β-catenin/VE-cadherin interaction at the cell membrane. This work shows that IQGAP1 is essential to ECs alignment under shear stress and that AJ remodeling represents one of the mechanisms involved. These results provide a new approach to understand ECs alignment under to shear stress.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0079919PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3838429PMC
September 2014

Doppler resistive index to reflect regulation of renal vascular tone during sepsis and acute kidney injury.

Crit Care 2012 Sep 12;16(5):R165. Epub 2012 Sep 12.

Introduction: Renal resistive index (RI), determined by Doppler ultrasonography, directly reveals and quantifies modifications in renal vascular resistance. The aim of this study was to evaluate if mean arterial pressure (MAP) is determinant of renal RI in septic, critically ill patients suffering or not from acute kidney injury (AKI).

Methods: This prospective observational study included 96 patients. AKI was defined according to RIFLE criteria and transient or persistent AKI according to renal recovery within 3 days.

Results: Median renal RIs were 0.72 (0.68-0.75) in patients without AKI and 0.76 (0.72-0.80) in patients with AKI (P = 0.001). RIs were 0.75 (0.72-0.79) in transient AKI and 0.77 (0.70-0.80) in persistent AKI (P = 0.84). RI did not differ in patients given norepinephrine infusion and was not correlated with norepinephrine dose. RI was correlated with MAP (ρ = -0.47; P = 0.002), PaO2/FiO2 ratio (ρ = -0.33; P = 0.04) and age (ρ = 0.35; P = 0.015) only in patients without AKI.

Conclusions: A poor correlation between renal RI and MAP, age, or PaO2/FiO2 ratio was found in septic and critically ill patients without AKI compared to patients with AKI. These findings suggest that determinants of RI are multiple. Renal circulatory response to sepsis estimated by Doppler ultrasonography cannot reliably be predicted simply from changes in systemic hemodynamics. As many factors influence its value, the interest in a single RI measurement at ICU admission to determine optimal MAP remains uncertain.
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http://dx.doi.org/10.1186/cc11517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682260PMC
September 2012

IQGAP1 interacts with components of the slit diaphragm complex in podocytes and is involved in podocyte migration and permeability in vitro.

PLoS One 2012 25;7(5):e37695. Epub 2012 May 25.

INSERM U1026, Université Bordeaux Segalen, Bordeaux, France.

IQGAP1 is a scaffold protein that interacts with proteins of the cytoskeleton and the intercellular adhesion complex. In podocytes, IQGAP1 is associated with nephrin in the glomerular slit diaphragm (SD) complex, but its role remains ill-defined. In this work, we investigated the interaction of IQGAP1 with the cytoskeleton and SD proteins in podocytes in culture, and its role in podocyte migration and permeability. Expression, localization, and interactions between IQGAP1 and SD or cytoskeletal proteins were determined in cultured human podocytes by Western blot (WB), immunocytolocalization (IC), immunoprecipitation (IP), and In situ Proximity Ligation assay (IsPL). Involvement of IQGAP1 in migration and permeability was also assessed. IQGAP1 expression in normal kidney biopsies was studied by immunohistochemistry. IQGAP1 expression by podocytes increased during their in vitro differentiation. IC, IP, and IsPL experiments showed colocalizations and/or interactions between IQGAP1 and SD proteins (nephrin, MAGI-1, CD2AP, NCK 1/2, podocin), podocalyxin, and cytoskeletal proteins (α-actinin-4). IQGAP1 silencing decreased podocyte migration and increased the permeability of a podocyte layer. Immunohistochemistry on normal human kidney confirmed IQGAP1 expression in podocytes and distal tubular epithelial cells and also showed an expression in glomerular parietal epithelial cells. In summary, our results suggest that IQGAP1, through its interaction with components of SD and cytoskeletal proteins, is involved in podocyte barrier properties.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037695PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3360763PMC
December 2012

A protective role for CD154 in hepatic steatosis in mice.

Hepatology 2010 Dec 9;52(6):1968-79. Epub 2010 Nov 9.

Inserm U889, National Institute for Health and Medical Research U889, Bordeaux University, F-33076 Bordeaux, France.

Unlabelled: Inflammation and lipid metabolism pathways are linked, and deregulation of this interface may be critical in hepatic steatosis. The importance of the dialog between inflammatory signaling pathways and the unfolded protein response (UPR) in metabolism has been underlined. Herein, we studied the role of CD154, a key mediator of inflammation, in hepatic steatosis. To this end, Balb/c mice, wild-type or deficient in CD154 (CD154KO), were fed a diet rich in olive oil. In vitro, the effect of CD154 was studied on primary hepatocyte cultures and hepatocyte-derived cell lines. Results showed that CD154KO mice fed a diet rich in olive oil developed hepatic steatosis associated with reduced apolipoprotein B100 (apoB100) expression and decreased secretion of very low-density lipoproteins. This phenotype correlated with an altered UPR as assessed by reduced X-Box binding protein-1 (XBP1) messenger RNA (mRNA) splicing and reduced phosphorylation of eukaryotic initiation factor 2α. Altered UPR signaling in livers of CD154KO mice was confirmed in tunicamycin (TM) challenge experiments. Treatment of primary hepatocyte cultures and hepatocyte-derived cell lines with soluble CD154 increased XBP1 mRNA splicing in cells subjected to either oleic acid (OA) or TM treatment. Moreover, CD154 reduced the inhibition of apoB100 secretion by HepG2 cells grown in the presence of high concentrations of OA, an effect suppressed by XBP1 mRNA silencing and in HepG2 cells expressing a dominant negative form of inositol requiring ER-to-nucleus signaling protein-1. The control of the UPR by CD154 may represent one of the mechanisms involved in the pathophysiology of hepatic steatosis.

Conclusion: Our study identifies CD154 as a new mediator of hepatic steatosis.
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http://dx.doi.org/10.1002/hep.23935DOI Listing
December 2010

Immunohistochemical study of the phenotypic change of the mesenchymal cells during portal tract maturation in normal and fibrous (ductal plate malformation) fetal liver.

Comp Hepatol 2009 Jul 14;8. Epub 2009 Jul 14.

INSERM U889, Université Bordeaux2, F-33076 Bordeaux, France.

Background: In adult liver, the mesenchymal cells, portal fibroblasts and vascular smooth muscle cells can transdifferentiate into myofibroblasts, and are involved in portal fibrosis. Differential expression of markers, such as alpha-smooth muscle actin (ASMA), h-caldesmon and cellular retinol-binding protein-1 allows their phenotypic discrimination. The aim of our study was to explore the phenotypic evolution of the mesenchymal cells during fetal development in normal liver and in liver with portal fibrosis secondary to ductal plate malformation in a series of Meckel-Gruber syndrome, autosomal recessive polycystic kidney disease and Ivemark's syndrome.

Results: At the early steps of the portal tract maturation, portal mesenchymal cells expressed only ASMA. During the maturation process, these cells were found condensed around the biliary and vascular structures. At the end of maturation process, only cells around vessels expressed ASMA and cells of the artery tunica media also expressed h-caldesmon. In contrast, ASMA positive cells persisted around the abnormal biliary ducts in fibrous livers.

Conclusion: As in adult liver, there is a phenotypic heterogeneity of the mesenchymal cells during fetal liver development. During portal tract maturation, myofibroblastic cells disappear in normal development but persist in fibrosis following ductal plate malformation.
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http://dx.doi.org/10.1186/1476-5926-8-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2721154PMC
July 2009

The transport of high amounts of vascular endothelial growth factor by blood platelets underlines their potential contribution in systemic sclerosis angiogenesis.

Rheumatology (Oxford) 2009 Sep 23;48(9):1036-44. Epub 2009 Jun 23.

Inserm U889, IFR 66, Bordeaux, France.

Objectives: Altered angiogenesis is a characteristic feature in SSc and remains ill-understood. VEGF is believed to play a central role. Serum VEGF is elevated in SSc patients but questions remain concerning the source of circulating VEGF. Here we investigated platelet activation and the role of platelets as a source of VEGF and other angiogenic mediators in this disease.

Methods: A cohort of 40 patients with SSc was included. Age- and sex-matched healthy subjects and subjects presenting a primary RP were included as controls. Platelets were isolated, activated with thrombin and the secretion of VEGF, platelet derived growth factor, homodimeric form BB (PDGF-BB), TGF-beta1 and angiopoietins-1 and -2 measured. Plasma concentrations of these mediators and the functionality of platelet-derived VEGF were also studied. Platelet activation was assayed by measuring plasma beta-thromboglobulin and expression of P-selectin on platelets. The effect of iloprost on VEGF secretion by platelets was studied.

Results: Platelets from SSc patients, in contrast to controls, secreted large amounts of VEGF when activated, but not PDGF-BB, TGF-beta1 or angiopoietins. Increased expression of membrane P-selectin confirmed platelet activation in the patients. Iloprost inhibited VEGF secretion by platelets both in vivo and in vitro, through inhibition of platelet activation.

Conclusions: Platelets transport high levels of VEGF in SSc. They may contribute to circulating VEGF because of ongoing activation in the course of the disease. If activated at the contact of injured endothelium, platelets may be important in the altered angiogenesis associated with the disease through the secretion of high levels of VEGF.
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http://dx.doi.org/10.1093/rheumatology/kep154DOI Listing
September 2009

Tissue inhibitors of matrix metalloproteinases in platelets and megakaryocytes: a novel organization for these secreted proteins.

Exp Hematol 2009 Jul 3;37(7):849-56. Epub 2009 May 3.

INSERM U889 and Université Victor Segalen Bordeaux 2, France.

Objective: Expression of tissue inhibitors of matrix metalloproteinases (TIMPs) is one way that activated platelets intervene in tissue remodeling and angiogenesis. Our study was designed to investigate their synthesis in megakaryocytes (MKs) and their storage in platelets.

Materials And Methods: TIMP expression in MKs derived from blood CD34(+) progenitor cells of normal donors and a megakaryocytic cell line (CHRF-288-11) grown in serum-free conditions and platelets from normal donors or two patients with gray platelet syndrome was studied by immunofluorescence labeling, reverse transcription-polymerase chain reaction, and western blotting.

Results: Biosynthesis of TIMPs 1-4 in MKs was indicated by presence of their messenger RNAs as shown by polymerase chain reaction and of their proteins. Immunofluorescence labeling suggested a primarily granular localization of TIMPs in MKs and platelets. But when colocalization with von Willebrand factor, fibrinogen, P-selectin, and other alpha-granule proteins was assessed in platelets by confocal microscopy, TIMP-1, -2, and -4 were localized as distinct fluorescent patches apart from the established alpha-granule markers and largely independent of platelet metalloproteinases. TIMP-3 differed for it also had an alpha-granule location. Western blotting confirmed the presence of TIMPs 1-4 in platelets and thrombin activation resulted in their extensive release to the medium. Platelets from two patients with gray platelet syndrome, congenitally deficient in alpha-granules, showed sparse labeling of von Willebrand factor and fibrinogen confined to vestigial alpha-granules; however, localization of the TIMPs was unchanged.

Conclusions: TIMPs are synthesized and organized in MKs and platelets independently of other secreted proteins present in alpha-granule pools.
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http://dx.doi.org/10.1016/j.exphem.2009.03.009DOI Listing
July 2009

Alterations in cytoskeletal protein expression by mycophenolic acid in human mesangial cells requires Rac inactivation.

Biochem Pharmacol 2007 May 28;73(9):1491-8. Epub 2006 Dec 28.

Inserm, E362 Bordeaux, France.

In response to glomerular injury, mesangial cells are activated into myofibroblasts, which contribute to the physiopathology of glomerulosclerosis. We have previously shown that chronic treatment of cultured human mesangial cells with mycophenolic acid (MPA), a specific inhibitor of guanosine nucleotide synthesis, prevents their activation and alters cytoskeleton protein expression and associated functions, such as contractility and migratory capacity. The aim of the present study was to explore the mechanisms underlying MPA-induced mesangial cytoskeleton alterations. We therein show that coincubation with guanosine (100 microM) compensates for the effects of MPA on mesangial cell proliferation and migration, and prevents MPA-induced overexpression of alpha-smooth muscle actin (SMA) and basic calponin (b-calp), indicating that guanylates are involved in mesangial responses to MPA. MPA decreased the GTP-bound (active) form of both RhoA, Rac1 and Cdc42, and specifically altered the expression level of Rac1. Pharmacological inhibition of RhoA activity reduced expression of both SMA and calponin, whereas overexpression of a dominant-negative form of Rac1 increased SMA expression. Conversely, overexpression of constitutively active Rac1 resulted in SMA and b-calp down-regulation, and fully prevented their stimulation by MPA, indicating that Rac inactivation is responsible for MPA effects on mesangial cytoskeletal expression. These results show that in human mesangial cells, RhoA and Rac1 exert opposite effects on the expression of two major cytoskeletal proteins: SMA and basic calponin. Moreover, these data highlight for the first time an integrated mechanism whereby MPA regulates mesangial phenotype, which is mediated by loss of Rac activity.
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http://dx.doi.org/10.1016/j.bcp.2006.12.025DOI Listing
May 2007

The ARE-associated factor AUF1 binds poly(A) in vitro in competition with PABP.

Biochem J 2006 Dec;400(2):337-47

INSERM, E362, Bordeaux, F-33076 France.

The ARE (AU-rich element) is a post-transcriptional element controlling both mRNA turnover and translation initiation by primarily inducing poly(A) tail shortening. The mechanisms by which the ARE-associated proteins induce deadenylation are still obscure. One possibility among others would be that an ARE-ARE-BP (ARE-binding protein) complex intervenes in the PABP [poly(A)-binding protein]-poly(A) tail association and facilitates poly(A) tail accessibility to deadenylases. Here, we show by several experimental approaches that AUF1 (AU-rich element RNA-binding protein 1)/hnRNP (heterogeneous nuclear ribonucleoprotein) D, an mRNA-destabilizing ARE-BP, can bind poly(A) sequence in vitro. First, endogenous AUF1 proteins from HeLa cells specifically bound poly(A), independently of PABP. Secondly, using polyadenylated RNA probes, we showed that (i) the four recombinant AUF1 isoforms bind poly(A) as efficiently as PABP, (ii) the AUF1 binding to poly(A) does not change when the polyadenylated probe contains the GM-CSF (granulocyte/macrophage-colony stimulating factor) ARE, suggesting that, in vitro, the AUF1-poly(A) association was independent of the ARE sequence itself. In vitro, the binding of AUF1 isoforms to poly(A) displayed oligomeric and co-operative properties and AUF1 efficiently displaced PABP from the poly(A). Finally, the AUF1 molar concentration in HeLa cytoplasm was only 2-fold lower than that of PABP, whereas in the nucleus, its molar concentration was similar to that of PABP. These in vitro results suggest that, in vivo, AUF1 could compete with PABP for the binding to poly(A). Altogether, our results may suggest a role for AUF1 in controlling PABP-poly(A) tail association.
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http://dx.doi.org/10.1042/BJ20060328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1652824PMC
December 2006

Activation of mesangial cells by platelets in systemic lupus erythematosus via a CD154-dependent induction of CD40.

Kidney Int 2005 Nov;68(5):2068-78

Département de Néphrologie, Hôpital Pellegrin, Bordeaux, France.

Background: Platelets are potential contributors to glomerular injury via the release of chemotactic and/or mitogenic mediators upon activation or through direct CD154/CD40-dependent interaction with cell components of the glomerulus. We examined whether platelets could activate mesangial cells and the potential role of the platelet-associated CD154.

Methods: Thrombin-activated platelets from systemic lupus erythematosus (SLE) patients or from disease or healthy controls were grown with human mesangial cells in the presence or not of a neutralizing anti-CD154 antibody either in contact or in a noncontact setting, the platelets and mesangial cells being separated by a pore size semipermeable membrane. The induction of mesangial cell surface antigens was assayed by flow cytometry. The quantification of mesangial cell proliferation was performed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and the production of transforming growth factor-beta1 (TGF-beta1), platelet-derived growth factor (PDGF) and soluble CD40 were measured by enzyme-linked immunosorbent assay (ELISA).

Results: Activated platelets from patients with SLE could induce an up-regulation of the expression of CD40 on mesangial cells with a concomitant release of soluble CD40. This induction required a direct contact between platelets and mesangial cells and was dependent upon the platelet-associated CD154. Pathologic consequences of the up-regulation of CD40 were a CD40-dependent stimulation of the proliferation of mesangial cells and a CD40-dependent increased production of TGF-beta1 by these cells.

Conclusion: Platelets from patients with SLE can activate mesangial cells through CD40/CD154 interactions, leading to an induction of proliferation of the mesangial cells and an enhanced production of TGF-beta1, a profibrotic cytokine.
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http://dx.doi.org/10.1111/j.1523-1755.2005.00663.xDOI Listing
November 2005

[Platelet-associated CD154: a new interface in haemostasis and in the inflammatory reaction].

Med Sci (Paris) 2005 Oct;21(10):825-31

GREF Inserm E362, IFR 66, Université de Bordeaux 2 et Département de Néphrologie, Hôpital Pellegrin, France.

Blood platelets play a crucial part in the blood clotting process by forming the platelet plug. Recent evidence indicates that they are likely to play a key role in the inflammatory reaction via CD154/CD40 interactions. CD40 was known to be widely expressed, for instance on cells of the vasculature including endothelial cells, smooth muscle cells and macrophages. It was also known that the triggering of CD40 on these cells led to the acquisition of an activated pro-inflammatory and pro-coagulant phenotype. It was subsequently shown that platelets express CD154 which is cryptic in unstimulated platelets but is expressed at the platelet surface upon platelet activation. When expressed at the platelet surface and exposed to CD40-expressing vascular cells, the platelet-associated CD154 triggers a variety of pro-inflammatory and pro-coagulant responses including induction of adhesion receptors, release of cytokines and chemokines, induction of tissue factor and of metalloproteinases. Platelet-associated CD154 is also involved in platelet/platelet interactions during platelet aggregation. Furthermore, in vivo models have emphasized the critical role of the platelet-associated CD154 in the progression of atherosclerotic disease and in the stabilization of arterial thrombi. Recent data show that CD40-bearing cells involved in fibrosis such as hepatic stellate cells and glomerular mesangial cells also respond to platelet-associated CD154, thus suggesting a new mechanism by which platelets may be instrumental in the inflammatory diseases of the liver or the kidney. Finally, platelet-associated CD154 has been shown to have immune competence both in vitro and in vivo, observations that open new fields of research on the potential implications of platelets in the immune response and auto-immune diseases.
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http://dx.doi.org/10.1051/medsci/20052110825DOI Listing
October 2005

In vivo MR imaging of intravascularly injected magnetically labeled mesenchymal stem cells in rat kidney and liver.

Radiology 2004 Dec 14;233(3):781-9. Epub 2004 Oct 14.

Laboratoire d'Imagerie Moléculaire et Fonctionnelle, Equipes de Recherche Technologique, Centre National de la Recherche Scientifique, Université Victor Segalen Bordeaux-2, 146 rue Leo Saignat, Case 117, 33076 Bordeaux, France.

Purpose: To evaluate in vivo magnetic resonance (MR) imaging with a conventional 1.5-T system for depiction and tracking of intravascularly injected superparamagnetic iron oxide (SPIO)-labeled mesenchymal stem cells (MSCs).

Materials And Methods: This study was conducted in accordance with French law governing animal research and met guidelines for animal care and use. Rat MSCs were labeled with SPIO and transfection agent. Relaxation rates at 1.5 T, cell viability, proliferation, differentiation capacity, and labeling stability were assessed in vitro as a function of SPIO concentration. MSCs were injected into renal arteries of healthy rats (labeled cells in four, unlabeled cells in two) and portal veins of rats treated with carbon tetrachloride to induce centrolobular liver necrosis (labeled cells and unlabeled cells in two each). Follow-up serial T2*-weighted gradient-echo MR imaging and R2* mapping were performed. MR imaging findings were compared histologically.

Results: SPIO labeling caused a strong R2* effect that increased linearly with iron dose; R2* increase for cells labeled for 48 hours with 50 microg of iron per milliliter was 50 sec(-1) per million cells per milliliter. R2* was proportional to iron load of cells. SPIO labeling did not affect cell viability (P > .27). Labeled cells were able to differentiate into adipocytes and osteocytes. Proliferation was substantially limited for MSCs labeled with 100 microg Fe/mL or greater. Label half-life was longer than 11 days. In normal kidneys, labeled MSCs caused signal intensity loss in renal cortex. After labeled MSC injection, diseased liver had diffuse granular appearance. Cells were detected for up to 7 days in kidney and 12 days in liver. Signal intensity loss and fading over time were confirmed with serial R2* mapping. At histologic analysis, signal intensity loss correlated with iron-loaded cells, primarily in renal glomeruli and hepatic sinusoids; immunohistochemical analysis results confirmed these cells were MSCs.

Conclusion: MR imaging can aid in monitoring of intravascularly administered SPIO-labeled MSCs in vivo in kidney and liver.
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http://dx.doi.org/10.1148/radiol.2333031714DOI Listing
December 2004

Platelet-associated CD154 in immune thrombocytopenic purpura.

Blood 2005 Jan 10;105(1):215-8. Epub 2004 Jun 10.

CNRS FRE 2617 and UMR 5540, Université de Bordeaux 2, France.

CD40-ligand (CD154) is expressed on activated CD4+ T lymphocytes and is essential for the T cell-dependent activation of B lymphocytes. CD154 is also expressed at the activated platelet surface. In this study, we show that platelet-associated CD154 is increased in immune thrombocytopenic purpura (ITP), a disease characterized by an autoimmune response against proteins of the platelet membrane. CD154 and its messenger RNA were also present in increased amounts in the megakaryocytes of patients with ITP. We found that platelet-associated CD154 is competent to induce the CD40-dependent proliferation of B lymphocytes, and we observed an in vitro CD154-dependent production of antibodies to the GPIIb/IIIa complex (integrin alphaIIbbeta3) when platelets and peripheral blood B lymphocytes from ITP patients with circulating anti-GPIIb/IIIa antibody were cultured together. Therefore, platelet-associated CD154 expression is increased in ITP and is able to drive the activation of autoreactive B lymphocytes in this disease.
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http://dx.doi.org/10.1182/blood-2003-07-2367DOI Listing
January 2005

In vivo studies of translational repression mediated by the granulocyte-macrophage colony-stimulating factor AU-rich element.

J Biol Chem 2004 Apr 15;279(14):13354-62. Epub 2004 Jan 15.

FRE 2617 CNRS, Université Victor Ségalen Bordeaux 2, 33076 Bordeaux, France.

The AU-rich element (ARE) controls the turnover of many unstable mRNAs and their translation. The granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE is known to be a destabilizing element, but its role in translation remains unclear. Here we studied in vivo the role of the GM-CSF ARE on the mRNA and protein expressions of an enhanced green fluorescent protein reporter gene. The GM-CSF ARE had a repressor effect on translation independently of its effect on mRNA levels. In the context of an internal ribosome entry site, the GM-CSF ARE still repressed translation but was no longer functional as a destabilizing element. Gel retardation assays showed that poly(A)-binding protein is displaced from the poly(A) tail when the ARE is present in the 3'-untranslated region. These data suggest that the GM-CSF ARE controls translation and mRNA decay by interfering with poly(A)-binding protein-mediated mRNA circularization.
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http://dx.doi.org/10.1074/jbc.M308003200DOI Listing
April 2004

Iron particle labeling of haematopoietic progenitor cells: an in vitro study.

Biosci Rep 2002 Oct-Dec;22(5-6):549-54

Laboratoire de Neurobiologie des Affections de la Myéline EA-2966, BP 78, Université Victor Ségalen, 33076 Bordeaux, France.

We present a method for labeling bone marrow haematopoietic progenitor cells with iron particles. Labeling was assessed by magnetic resonance imaging and electron microscopy. Labeling with iron particles could allow the following by imaging techniques of haematopoietic cells in physiologic and pathologic conditions such as the engraftment of haematopoietic progenitor cells or the migration of myelomonocytic cells in inflammatory diseases.
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http://dx.doi.org/10.1023/a:1022030004597DOI Listing
September 2003

Flt3-ligand induces adhesion of haematopoietic progenitor cells via a very late antigen (VLA)-4- and VLA-5-dependent mechanism.

Br J Haematol 2003 Mar;120(5):782-6

UMR 5540, Université Victor Ségalen Bordeaux 2, Bordeaux, France.

The adhesion of haematopoietic progenitor cells (HPC) to the bone marrow microenvironment is a process regulated by cytokines. In this study, we have shown that flt3-ligand (FL), a growth factor that controls early haematopoiesis, regulated the function and expression of the beta-1 integrins, very late antigen (VLA)-4 and VLA-5 on HPC. The modulation of the adhesiveness of HPC by FL was studied by adhesion assays on umbilical vein endothelial cells (HUVEC). Stimulation by FL induced two peaks of increased adhesiveness of HPC. The first peak was at around 30 min and was mechanistically related to an activation of the beta-1 integrins, mainly VLA-4 and VLA-5. The second peak was at around 12 h and was related to increased expression of VLA-4 and VLA-5. The control of HPC adhesiveness by FL is a previously unreported property of FL that may be important for the homing and the retention of flt3-expressing HPC within the bone marrow microenvironment.
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http://dx.doi.org/10.1046/j.1365-2141.2003.04155.xDOI Listing
March 2003