Publications by authors named "Damien Schapman"

18 Publications

  • Page 1 of 1

Comparative Structural and Functional Analyses of the Fusiform, Oval, and Triradiate Morphotypes of Pt3 Strain.

Front Plant Sci 2021 12;12:638181. Epub 2021 Apr 12.

Normandie University, UNIROUEN, Laboratoire Glycobiologie et Matrice Extracellulaire Végétale (Glyco-MEV) EA4358, Rouen, France.

The diatom is a marine unicellular microalga that exists under three main morphotypes: oval, fusiform, and triradiate. Previous works have demonstrated that the oval morphotype of Pt3 strain presents specific metabolic features. Here, we compared the cellular organization of the main morphotypes of the diatom Pt3 strain through transmission electron and advanced light microscopies. The three morphotypes share similarities including spectral characteristics of the plastid, the location of the nucleus, the organization of mitochondria around the plastid as well as the existence of both a F-actin cortex, and an intracellular network of F-actin. In contrast, compared to fusiform and triradiate cells, oval cells spontaneously release proteins more rapidly. In addition, comparison of whole transcriptomes of oval versus fusiform or triradiate cells revealed numerous differential expression of positive and negative regulators belonging to the complex dynamic secretory machinery. This study highlights the specificities occurring within the oval morphotype underlying that the oval cells secrete proteins more rapidly.
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http://dx.doi.org/10.3389/fpls.2021.638181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8072121PMC
April 2021

IHC_Tool: An open-source Fiji procedure for quantitative evaluation of cross sections of testicular explants.

Reprod Biol 2021 Apr 24;21(2):100507. Epub 2021 Apr 24.

Normandie Univ, UNIROUEN, EA 4308 "Gametogenesis and Gamete Quality", Rouen University Hospital, Department of Reproductive Biology - CECOS, F 76000, Rouen, France; Institute for Research and Innovation in Biomedicine (IRIB), Rouen, France.

Immunohistochemical analysis is a routine procedure for clinical and research studies in male fertility. However, most of the interpretations remain subjective and time-consuming, with inherent intra- and inter-observer variability. Given the prognostic and research implications of testicular assessment, a more objective and less time-consuming method is required. In the current study, we used in vitro matured pre-pubertal murine testes as a model. The main objective was to develop an affordable automated digital immunohistochemistry image analysis tool for an unbiased and quantitative assessment of testicular tissue sections. Testicular explants were fixed, cut, and stained for specific germ cell markers. The classical manual counting procedure was evaluated. Background and noise were reduced on brightfield images. Photomicrographs were stitched (Background_Elimination_Stitching) to create high-quality images. Two procedures were evaluated (IHC_Tool and Stained_Nuclear_Area); then a procedure (Necrotic_Area_Elimination) allowing withdrawal of the necrotic area observed after culture was assessed. Finally, the number of stained nuclei in the unaltered tissue area was extracted. The automated IHC_Tool procedure with images saved as TIFF at a ×200 magnification allowed the most rigorous cell quantification. IHC_Tool developed for testicular sample analysis can be used for various types of tissues. We foresee that this method will minimize inter-observer variations across laboratories and will be helpful for clinical trials and translational initiatives.
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http://dx.doi.org/10.1016/j.repbio.2021.100507DOI Listing
April 2021

3-Benzoylquinoxalinone as a photoaffinity labelling derivative with fluorogenic properties allowing reaction monitoring under "no-wash" conditions.

Chem Commun (Camb) 2021 Apr 23;57(32):3893-3896. Epub 2021 Mar 23.

Normandie Univ, CNRS, UNIROUEN, INSA Rouen, COBRA (UMR 6014), Rouen 76000, France.

Described herein is a quinoxalinone-based photoaffinity probe with caged fluorescence properties. Upon visible blue LED irradiation (λ 450 nm), this photo-crosslinker is able to covalently capture proteins with concomitant fluorescence labelling. This process enables monitoring applications under "no wash" conditions.
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http://dx.doi.org/10.1039/d1cc01072gDOI Listing
April 2021

Antibody-Conjugated Nanocarriers for Targeted Antibiotic Delivery: Application in the Treatment of Bacterial Biofilms.

Biomacromolecules 2021 Apr 12;22(4):1639-1653. Epub 2021 Mar 12.

Normandie Université, UNIROUEN, INSA Rouen, CNRS, PBS, 76000 Rouen, France.

Conventional antibiotic treatment is in most cases insufficient to eradicate biofilm-related infections, resulting in high risk of treatment failure and recurrent infections. Recent studies have shown that novel methods of antibiotic delivery can improve clinical outcomes and reduce the emergence of antibiotic resistance. The objectives of this work were to develop and evaluate a targeting nanocarrier system that enables effective delivery of antimicrobial drugs to , a commonly virulent human pathogen. For this purpose, we first prepared a formulation of polymeric nanoparticles (NPs) suitable for encapsulation and sustained release of antibiotics. A specific antibody against was used as a targeting ligand and was covalently immobilized onto the surface of nanoparticulate materials. It was demonstrated that the targeting NPs preferentially bound cells and presented an elevated accumulation in the biofilm. Compared to free-form antibiotic, the antibiotic-loaded targeting NPs significantly enhanced bactericidal activity against both in planktonic and biofilm forms. Using a mouse infection model, we observed improved therapeutic efficacy of these antibiotic-loaded NPs after a single intravenous administration. Taken together, our studies show that the targeting nanoparticulate system could be a promising strategy to enhance the biodistribution of antibiotics and thereby improve their efficacy.
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http://dx.doi.org/10.1021/acs.biomac.1c00082DOI Listing
April 2021

Root Border Cells and Mucilage Secretions of Soybean, (Merr) L.: Characterization and Role in Interactions with the Oomycete .

Cells 2020 09 30;9(10). Epub 2020 Sep 30.

Laboratoire de Glycobiologie et Matrice Extracellulaire Végétale, UPRES-EA 4358, Fédération de Recherche « Normandie-Végétal »-FED 4277, Université de ROUEN Normandie, UFR des Sciences et Techniques, F-76821 Mont-Saint-Aignan, France.

Root border cells (BCs) and their associated secretions form a protective structure termed the root extracellular trap (RET) that plays a major role in root interactions with soil borne microorganisms. In this study, we investigated the release and morphology of BCs of using light and cryo-scanning electron microscopy (SEM). We also examined the occurrence of cell-wall glycomolecules in BCs and secreted mucilage using immunofluorescence microscopy in conjunction with anti-glycan antibodies. Our data show that root tips released three populations of BCs defined as spherical, intermediate and elongated cells. The mechanism of shedding seemed to be cell morphotype-specific. The data also show that mucilage contained pectin, cellulose, extracellular DNA, histones and two hemicellulosic polysaccharides, xyloglucan and heteromannan. The latter has never been reported previously in any plant root secretions. Both hemicellulosic polysaccharides formed a dense fibrillary network embedding BCs and holding them together within the mucilage. Finally, we investigated the effect of the RET on the interactions of root with the pathogenic oomycete early during infection. Our findings reveal that the RET prevented zoospores from colonizing root tips by blocking their entry into root tissues and inducing their lysis.
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http://dx.doi.org/10.3390/cells9102215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7650559PMC
September 2020

Lymphatic and Immune Cell Cross-Talk Regulates Cardiac Recovery After Experimental Myocardial Infarction.

Arterioscler Thromb Vasc Biol 2020 07 14;40(7):1722-1737. Epub 2020 May 14.

From the Normandy University, UniRouen, Inserm (Institut National de la Santé et de la Recherche Médicale) UMR1096 (EnVI Laboratory), FHU REMOD-VHF, Rouen, France (H.M., A.D., V.T., I.B., J.P.H., S.R., J.R., S.F., V.R., P.M.).

Objective: Lymphatics play an essential pathophysiological role in promoting fluid and immune cell tissue clearance. Conversely, immune cells may influence lymphatic function and remodeling. Recently, cardiac lymphangiogenesis has been proposed as a therapeutic target to prevent heart failure after myocardial infarction (MI). We investigated the effects of gene therapy to modulate cardiac lymphangiogenesis post-MI in rodents. Second, we determined the impact of cardiac-infiltrating T cells on lymphatic remodeling in the heart. Approach and Results: Comparing adenoviral versus adeno-associated viral gene delivery in mice, we found that only sustained VEGF (vascular endothelial growth factor)-C therapy, achieved by adeno-associated viral vectors, increased cardiac lymphangiogenesis, and led to reduced cardiac inflammation and dysfunction by 3 weeks post-MI. Conversely, inhibition of VEGF-C/-D signaling, through adeno-associated viral delivery of soluble VEGFR3 (vascular endothelial growth factor receptor 3), limited infarct lymphangiogenesis. Unexpectedly, this treatment improved cardiac function post-MI in both mice and rats, linked to reduced infarct thinning due to acute suppression of T-cell infiltration. Finally, using pharmacological, genetic, and antibody-mediated prevention of cardiac T-cell recruitment in mice, we discovered that both CD4 and CD8 T cells potently suppress, in part through interferon-γ, cardiac lymphangiogenesis post-MI.

Conclusions: We show that resolution of cardiac inflammation after MI may be accelerated by therapeutic lymphangiogenesis based on adeno-associated viral gene delivery of VEGF-C. Conversely, our work uncovers a major negative role of cardiac-recruited T cells on lymphatic remodeling. Our results give new insight into the interconnection between immune cells and lymphatics in orchestration of cardiac repair after injury.
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http://dx.doi.org/10.1161/ATVBAHA.120.314370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310303PMC
July 2020

Investigating Tunneling Nanotubes in Cancer Cells: Guidelines for Structural and Functional Studies through Cell Imaging.

Biomed Res Int 2020 13;2020:2701345. Epub 2020 Apr 13.

Normandie University, UNIROUEN, INSERM, PRIMACEN, F-76821 Mont-Saint-Aignan, France.

By allowing insured communication between cancer cells themselves and with the neighboring stromal cells, tunneling nanotubes (TNTs) are involved in the multistep process of cancer development from tumorigenesis to the treatment resistance. However, despite their critical role in the biology of cancer, the study of the TNTs has been announced challenging due to not only the absence of a specific biomarker but also the fragile and transitory nature of their structure and the fact that they are hovering freely above the substratum. Here, we proposed to review guidelines to follow for studying the structure and functionality of TNTs in tumoral neuroendocrine cells (PC12) and nontumorigenic human bronchial epithelial cells (HBEC-3, H28). In particular, we reported how crucial is it (i) to consider the culture conditions (culture surface, cell density), (ii) to visualize the formation of TNTs in living cells (mechanisms of formation, 3D representation), and (iii) to identify the cytoskeleton components and the associated elements (categories, origin, tip, and formation/transport) in the TNTs. We also focused on the input of high-resolution cell imaging approaches including Stimulated Emission Depletion (STED) nanoscopy, Transmitted and Scanning Electron Microscopies (TEM and SEM). In addition, we underlined the important role of the organelles in the mechanisms of TNT formation and transfer between the cancer cells. Finally, new biological models for the identification of the TNTs between cancer cells and stromal cells (liquid air interface, , ) and the clinical considerations will also be discussed.
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http://dx.doi.org/10.1155/2020/2701345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7174938PMC
February 2021

Fluorophore-Assisted Click Chemistry through Copper(I) Complexation.

Biomolecules 2020 04 16;10(4). Epub 2020 Apr 16.

Normandie Univ, CNRS, UNIROUEN, INSA Rouen, COBRA (UMR 6014), 76000 Rouen, France.

The copper-catalyzed alkyne-azide cycloaddition (CuAAC) is one of the most powerful chemical strategies for selective fluorescent labeling of biomolecules in in vitro or biological systems. In order to accelerate the ligation process and ensure efficient formation of conjugates under diluted conditions, external copper(I) ligands or sophisticated copper(I)-chelating azides are used. This latter strategy, however, increases the bulkiness of the triazole linkage, thus perturbing the biological function or dynamic behavior of the conjugates. In a proof-of-concept study, we investigated the use of an extremely compact fluorophore-based copper(I) chelating azide in order to accelerate the CuAAC with concomitant fluorescence labeling; in our strategy, the fluorophore is able to complex copper(I) species while retaining its photophysical properties. It is believed that this unprecedented approach which was applied for the labeling of a short peptide molecule and the fluorescent labeling of live cells, could be extended to other families of nitrogen-based fluorophores in order to tune both the reaction rate and photophysical characteristics.
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http://dx.doi.org/10.3390/biom10040619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225998PMC
April 2020

Chromogranin A preferential interaction with Golgi phosphatidic acid induces membrane deformation and contributes to secretory granule biogenesis.

FASEB J 2020 05 29;34(5):6769-6790. Epub 2020 Mar 29.

Laboratoire de Différenciation et Communication Neuronale et Neuroendocrine, Institut de Recherche et d'Innovation Biomédicale de Normandie, Normandie University, UNIROUEN, INSERM, U1239, Rouen, France.

Chromogranin A (CgA) is a key luminal actor of secretory granule biogenesis at the trans-Golgi network (TGN) level but the molecular mechanisms involved remain obscure. Here, we investigated the possibility that CgA acts synergistically with specific membrane lipids to trigger secretory granule formation. We show that CgA preferentially interacts with the anionic glycerophospholipid phosphatidic acid (PA). In accordance, bioinformatic analysis predicted a PA-binding domain (PABD) in CgA sequence that effectively bound PA (36:1) or PA (40:6) in membrane models. We identified PA (36:1) and PA (40:6) as predominant species in Golgi and granule membranes of secretory cells, and we found that CgA interaction with these PA species promotes artificial membrane deformation and remodeling. Furthermore, we demonstrated that disruption of either CgA PABD or phospholipase D (PLD) activity significantly alters secretory granule formation in secretory cells. Our findings show for the first time the ability of CgA to interact with PLD-generated PA, which allows membrane remodeling and curvature, key processes necessary to initiate secretory granule budding.
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http://dx.doi.org/10.1096/fj.202000074RDOI Listing
May 2020

A versatile and recyclable molecularly imprinted polymer as an oxidative catalyst of sulfur derivatives: a new possible method for mustard gas and V nerve agent decontamination.

Chem Commun (Camb) 2019 Oct;55(88):13243-13246

Normandie Univ., INSA Rouen, UNIROUEN, CNRS, COBRA (UMR 6014 & FR3038), 76000 Rouen, France.

A molecularly imprinted polymer containing a porphyrin unit was developed as a biomimetic heterogenous catalyst for the oxidation of sulfur derivatives. Its catalytic efficiency under mild conditions and its easy recovery represent a great asset for the design of new decontamination tools for yperite and VX.
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http://dx.doi.org/10.1039/c9cc04928bDOI Listing
October 2019

Two-Photon Absorption and Cell Imaging of Fluorene-Functionalized Epicocconone Analogues.

Chemistry 2019 Aug 25;25(46):10954-10964. Epub 2019 Jul 25.

COBRA (UMR 6014 and FR 3038), Normandie Univ, CNRS, INSA Rouen, UNIROUEN, 76000, Rouen, France.

Epicocconone 1 is a natural chromophore isolated from the fungus Epicoccum nigrum that has shown applications in proteomics and fluorescent microscopy thanks to its unique pro-fluorescence properties. The modification of the skeleton of the natural product by replacing the triene side chain by a fluorenyl scaffold can noticeably increase the fluorophore's absorption coefficient. The synthesis of the analogues of the natural product has been made possible by the use of a palladium-catalyzed carbonylation reaction, allowing the construction of the β-keto-dioxinone key intermediate. Two-photon absorption cross-section measurements of the fluorenyl epicocconone analogues show a structure dependency with values ranging from 60 to 280 GM and live cell imaging show intense staining of intracellular vesicle-like structures around the nucleus.
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http://dx.doi.org/10.1002/chem.201902731DOI Listing
August 2019

Hydroxychloroquine reverses the prothrombotic state in a mouse model of antiphospholipid syndrome: Role of reduced inflammation and endothelial dysfunction.

PLoS One 2019 14;14(3):e0212614. Epub 2019 Mar 14.

Rouen University Hospital, Department of Internal Medicine, Rouen, France.

Antiphospholipid antibodies (aPL) promote endothelial dysfunction, inflammation and procoagulant state. We investigated the effect of hydroxychloroquine (HCQ) on prothrombotic state and endothelial function in mice and in human aortic endothelial cells (HAEC). Human aPL were injected to C57BL/6 mice treated or not with HCQ. Vascular endothelial function and eNOS were assessed in isolated mesenteric arteries. Thrombosis was assessed both in vitro by measuring thrombin generation time (TGT) and tissue factor (TF) expression and in vivo by the measurement of the time to occlusion in carotid and the total thrombosis area in mesenteric arteries. TGT, TF, and VCAM1 expression were evaluated in HAEC. aPL increased VCAM-1 expression and reduced endothelium dependent relaxation to acetylcholine. In parallel, aPL shortened the time to occlusion and extended thrombus area in mice. This was associated with an overexpression of TF and an increased TGT in mice and in HAEC. HCQ reduced clot formation as well as TGT, and improved endothelial-dependent relaxations. Finally, HCQ increased the p-eNOS/eNOS ratio. This study provides new evidence that HCQ improves procoagulant status and vascular function in APS by modulating eNOS, leading to an improvement in the production of NO.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0212614PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6417644PMC
November 2019

A role for RASSF1A in tunneling nanotube formation between cells through GEFH1/Rab11 pathway control.

Cell Commun Signal 2018 10 11;16(1):66. Epub 2018 Oct 11.

Normandie Univ, UNICAEN, CEA, CNRS, ISTCT/CERVOxy group, GIP CYCERON, F-14000, Caen, France.

Background: By allowing intercellular communication between cells, tunneling nanotubes (TNTs) could play critical role in cancer progression. If TNT formation is known to require cytoskeleton remodeling, key mechanism controlling their formation remains poorly understood.

Methods: The cells of human bronchial (HBEC-3, A549) or mesothelial (H2452, H28) lines are transfected with different siRNAs (inactive, anti-RASSF1A, anti-GEFH1 and / or anti-Rab11). At 48 h post-transfection, i) the number and length of the nanotubes per cell are quantified, ii) the organelles, previously labeled with specific tracers, exchanged via these structures are monitored in real time between cells cultured in 2D or 3D and in normoxia, hypoxia or in serum deprivation condition.

Results: We report that RASSF1A, a key-regulator of cytoskeleton encoded by a tumor-suppressor gene on 3p chromosome, is involved in TNTs formation in bronchial and pleural cells since controlling proper activity of RhoB guanine nucleotide exchange factor, GEF-H1. Indeed, the GEF-H1 inactivation induced by RASSF1A silencing, leads to Rab11 accumulation and subsequent exosome releasing, which in turn contribute to TNTs formation. Finally, we provide evidence involving TNT formation in bronchial carcinogenesis, by reporting that hypoxia or nutriment privation, two almost universal conditions in human cancers, fail to prevent TNTs induced by the oncogenic RASSF1A loss of expression.

Conclusions: This finding suggests for the first time that loss of RASSF1A expression could be a potential biomarker for TNTs formation, such TNTs facilitating intercellular communication favoring multistep progression of bronchial epithelial cells toward overt malignancy.
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http://dx.doi.org/10.1186/s12964-018-0276-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180646PMC
October 2018

Postnatal Migration of Cerebellar Interneurons.

Brain Sci 2017 Jun 6;7(6). Epub 2017 Jun 6.

Department of Neuroscience, School of Medicine, Yale University, New Haven, CT 06510, USA.

Due to its continuing development after birth, the cerebellum represents a unique model for studying the postnatal orchestration of interneuron migration. The combination of fluorescent labeling and ex/in vivo imaging revealed a cellular highway network within cerebellar cortical layers (the external granular layer, the molecular layer, the Purkinje cell layer, and the internal granular layer). During the first two postnatal weeks, saltatory movements, transient stop phases, cell-cell interaction/contact, and degradation of the extracellular matrix mark out the route of cerebellar interneurons, notably granule cells and basket/stellate cells, to their final location. In addition, cortical-layer specific regulatory factors such as neuropeptides (pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin) or proteins (tissue-type plasminogen activator (tPA), insulin growth factor-1 (IGF-1)) have been shown to inhibit or stimulate the migratory process of interneurons. These factors show further complexity because somatostatin, PACAP, or tPA have opposite or no effect on interneuron migration depending on which layer or cell type they act upon. External factors originating from environmental conditions (light stimuli, pollutants), nutrients or drug of abuse (alcohol) also alter normal cell migration, leading to cerebellar disorders.
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http://dx.doi.org/10.3390/brainsci7060062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5483635PMC
June 2017

Selective Stimulation of Cardiac Lymphangiogenesis Reduces Myocardial Edema and Fibrosis Leading to Improved Cardiac Function Following Myocardial Infarction.

Circulation 2016 Apr 1;133(15):1484-97; discussion 1497. Epub 2016 Mar 1.

From Inserm (Institut National de la Santé et de la Recherche Médicale) U1096, Rouen, France (O.H., C.P., M.H., L.N., J.-P.H., A.D., I.B., S.B., C.T., V.R., P.M., E.B.); Normandy University & University of Rouen, Institute for Research and Innovation in Biomedicine, France (O.H., C.P., M.H., L.G., L.N., J.-P.H., A.D., I.B., S.B., D.S., C.T., V.R., P.M., E.B.); PRIMACEN, Cell Imaging Platform of Normandy, Inserm, Mont-Saint-Aignan, France (L.G., D.S.); PICTUR, In Vivo Imaging Platform, University of Rouen, Institute for Research and Innovation in Biomedicine, France (L.N., C.T., P.M.); Reims Institute of Molecular Chemistry, UMR 7312 CNRS-URCA, University of Reims Champagne Ardenne, France (F.E.-L,); and Rouen University Hospital, Department of Pharmacology, France (C.T.).

Background: The lymphatic system regulates interstitial tissue fluid balance, and lymphatic malfunction causes edema. The heart has an extensive lymphatic network displaying a dynamic range of lymph flow in physiology. Myocardial edema occurs in many cardiovascular diseases, eg, myocardial infarction (MI) and chronic heart failure, suggesting that cardiac lymphatic transport may be insufficient in pathology. Here, we investigate in rats the impact of MI and subsequent chronic heart failure on the cardiac lymphatic network. Further, we evaluate for the first time the functional effects of selective therapeutic stimulation of cardiac lymphangiogenesis post-MI.

Methods And Results: We investigated cardiac lymphatic structure and function in rats with MI induced by either temporary occlusion (n=160) or permanent ligation (n=100) of the left coronary artery. Although MI induced robust, intramyocardial capillary lymphangiogenesis, adverse remodeling of epicardial precollector and collector lymphatics occurred, leading to reduced cardiac lymphatic transport capacity. Consequently, myocardial edema persisted for several months post-MI, extending from the infarct to noninfarcted myocardium. Intramyocardial-targeted delivery of the vascular endothelial growth factor receptor 3-selective designer protein VEGF-CC152S, using albumin-alginate microparticles, accelerated cardiac lymphangiogenesis in a dose-dependent manner and limited precollector remodeling post-MI. As a result, myocardial fluid balance was improved, and cardiac inflammation, fibrosis, and dysfunction were attenuated.

Conclusions: We show that, despite the endogenous cardiac lymphangiogenic response post-MI, the remodeling and dysfunction of collecting ducts contribute to the development of chronic myocardial edema and inflammation-aggravating cardiac fibrosis and dysfunction. Moreover, our data reveal that therapeutic lymphangiogenesis may be a promising new approach for the treatment of cardiovascular diseases.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.115.020143DOI Listing
April 2016

Plant cell wall imaging by metabolic click-mediated labelling of rhamnogalacturonan II using azido 3-deoxy-D-manno-oct-2-ulosonic acid.

Plant J 2016 Feb;85(3):437-47

Laboratoire Glycobiologie et Matrice Extracellulaire Végétale (Glyco-MEV), EA 4358, IRIB, VASI, Normandie Université, 76821, Mont-Saint-Aignan, France.

In plants, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) is a monosaccharide that is only found in the cell wall pectin, rhamnogalacturonan-II (RG-II). Incubation of 4-day-old light-grown Arabidopsis seedlings or tobacco BY-2 cells with 8-azido 8-deoxy Kdo (Kdo-N3 ) followed by coupling to an alkyne-containing fluorescent probe resulted in the specific in muro labelling of RG-II through a copper-catalysed azide-alkyne cycloaddition reaction. CMP-Kdo synthetase inhibition and competition assays showing that Kdo and D-Ara, a precursor of Kdo, but not L-Ara, inhibit incorporation of Kdo-N3 demonstrated that incorporation of Kdo-N3 occurs in RG-II through the endogenous biosynthetic machinery of the cell. Co-localisation of Kdo-N3 labelling with the cellulose-binding dye calcofluor white demonstrated that RG-II exists throughout the primary cell wall. Additionally, after incubating plants with Kdo-N3 and an alkynated derivative of L-fucose that incorporates into rhamnogalacturonan I, co-localised fluorescence was observed in the cell wall in the elongation zone of the root. Finally, pulse labelling experiments demonstrated that metabolic click-mediated labelling with Kdo-N3 provides an efficient method to study the synthesis and redistribution of RG-II during root growth.
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http://dx.doi.org/10.1111/tpj.13104DOI Listing
February 2016

Structural and functional analysis of tunneling nanotubes (TnTs) using gCW STED and gconfocal approaches.

Biol Cell 2015 Nov 17;107(11):419-25. Epub 2015 Jul 17.

Cell imaging platform of Normandy (PRIMACEN), Infrastructure en Biologie, Santé et Agronomie (IBiSA), Institut National de la Santé et de la Recherche Médicale (Inserm), Mont-Saint-Aignan, France.

Background Information: Tunneling nanotubes (TnTs) are thin plasma membrane bridges mediating transfers of materials and signals between cells. Heterogeneity of heterocellular and homocellular TnTs is largely described but ultrafine imaging of these light-sensitive floating nanometric structures represents a real challenge in microscopy. We propose here imaging strategies designed to dissect structural and dynamic aspects of TnT formation and function in fixed or living PC12 cells.

Results: Through time-gated Continuous Wave STimulated Emission Depletion (gCW STED) nanoscopy associated with deconvolution, we provided nanoscale details of membrane and cytoskeleton organisations in two subtypes of TnTs, namely type 1 TnT (TnT1) and type 2 TnT (TnT2). In fixed PC12 cells, TnT1 (length, several tens of micrometres; diameter, 100-650 nm) exhibited a large trumpet-shaped origin, a clear cytosolic tunnel and different bud-shaped connections from closed-ended to open-ended tips. TnT1 contained both actin and tubulin. TnT2 (length, max 20 μm, diameter, 70-200 nm) only contained actin without clear cytosolic tunnel. In living PC12 cells, we observed through gCW STED additional details, unrevealed so far, including a filament spindle emerging from an organising centre at the origin of TnT1 and branched or bulbous attachments of TnT2. However, the power of depletion laser in STED nanoscopy was deleterious for TnTs and prolonged time-lapse experiments were almost prohibited. By circumventing the hazard of photoxicity, we were able to monitor dynamics of bud-shaped tips and intercellular transfer of wheat germ agglutinin labelled cellular elements through time-gated confocal microscopy.

Conclusions: Our work identified new structural characteristics of two subtypes of TnTs in PC12 cells as well as dynamics of formation and transfer through complementary imaging methods combined with image processing. Therefore, we could achieve maximum lateral resolution and sample preservation during acquisitions to reveal new insights into TnT studies.

Significance: Due to large disparity of TnT-like structures in neuronal, immune, cancer or epithelial cells, high- and superresolution approaches can be utilised for full characterisation of these yet poorly understood routes of cell-to-cell communication.
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http://dx.doi.org/10.1111/boc.201500004DOI Listing
November 2015

Arteriogenic therapy by intramyocardial sustained delivery of a novel growth factor combination prevents chronic heart failure.

Circulation 2011 Aug 8;124(9):1059-69. Epub 2011 Aug 8.

Inserm U644, Institute for Biomedical Research, Rouen University, 22 Blvd Gambetta, 76183 Rouen, France.

Background: Therapeutic angiogenesis is a promising approach for the treatment of cardiovascular diseases, including myocardial infarction and chronic heart failure. We aimed to improve proangiogenic therapies by identifying novel arteriogenic growth factor combinations, developing injectable delivery systems for spatiotemporally controlled growth factor release, and evaluating functional consequences of targeted intramyocardial growth factor delivery in chronic heart failure.

Methods And Results: First, we observed that fibroblast growth factor and hepatocyte growth factor synergistically stimulate vascular cell migration and proliferation in vitro. Using 2 in vivo angiogenesis assays (n=5 mice per group), we found that the growth factor combination results in a more potent and durable angiogenic response than either growth factor used alone. Furthermore, we determined that the molecular mechanisms involve potentiation of Akt and mitogen-activated protein kinase signal transduction pathways, as well as upregulation of angiogenic growth factor receptors. Next, we developed crosslinked albumin-alginate microcapsules that sequentially release fibroblast growth factor-2 and hepatocyte growth factor. Finally, in a rat model of chronic heart failure induced by coronary ligation (n=14 to 15 rats per group), we found that intramyocardial slow release of fibroblast growth factor-2 with hepatocyte growth factor potently stimulates angiogenesis and arteriogenesis and prevents cardiac hypertrophy and fibrosis, as determined by immunohistochemistry, leading to improved cardiac perfusion after 3 months, as shown by magnetic resonance imaging. These multiple beneficial effects resulted in reduced adverse cardiac remodeling and improved left ventricular function, as revealed by echocardiography.

Conclusion: Our data showing the selective advantage of using fibroblast growth factor-2 together with hepatocyte growth factor suggest that this growth factor combination may constitute an efficient novel treatment for chronic heart failure.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.110.010264DOI Listing
August 2011