Höchstauflösendes Fluoreszenzmikroskop
Final Report Abstract
Since 01.12.2014 the confocal (CLSM) and stimulated emission depletion (STED) microscope is part of the IPEK optical imaging core facility of atherosclerosis which is located in the Klinikum der LMU Munich. In a collaborative approach, the core facility provides imaging technology and knowledge to internal (IPEK) and external (LMU and others) scientists. The CLSM/STED is mainly used for cardiovascular and immunological experiments that require high resolution multi target imaging of viable and fixed in vitro samples or histological slides. Furthermore, we aim to develop experimental methods for application of STED in the field of atherosclerosis. Finally, an important role for the core facility and as such the system is to educate and train young and established scientists to make use of optical imaging technology for their current and future scientific projects. The CLSM/STED system in combination with image deconvolution has been pivotal for a series of studies conducted at IPEK, both in confocal and STED mode: We have assessed platelet inhibition of collagen fibers, platelet glycoprotein (GPVI) and (non) cross-linked dimeric soluble GPVI-Fc (Revacept) binding to collagen fibers and fibrin. The nature of the targets (platelets, thin collagen and fibrin fibers) and the assessment of platelet adhesion inhibition by GPVI-Fc required high resolution, multicolor imaging by confocal microscopy and nanoscopic STED. The work has contributed to several publications. Confocal laser scanning microscopy was utilized to visualize and quantify endothelial permeability in (dissected) aortic arches in a study determining the effect of chronic Intake of the selective serotonin inhibitor fluoxetine on atherosclerosis. To contribute to a study on Chemokine interactome mapping for enabling tailored intervention in acute and chronic inflammation we have used a combination of confocal and STED imaging to visually reveal interactions between murine CCL5, CCL17, CCR4, and CCR5 on the surface of adherent DCs using a proximity ligation assay. Confocal/STED was used to reveal HNP1 and CCL5 (interactions) on vascular endothelium cells and its effects on monocyte adhesion. CD70 expression in human atheroma and by plaque resident macrophages in the murine aorta was revealed using multicolor confocal microscopy and subsequent image deconvolution. For a project studying CD27 co-stimulation and the abundancy of regulatory T cells in atherosclerosis in hyperlipidaemic mice, we have conducted immunohistochemistry and multichannel confocal microscopy to reveal the presence of CD27 and several other targets in murine aortic root sections.
Publications
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Optical imaging innovations for atherosclerosis research: multiphoton microscopy and optical nanoscopy. Arterioscler Thromb Vasc Biol. 2015;35(6):1339-46
Megens RTA, Bianchini M, Schmitt MNN, Weber C
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Recruitment of classical monocytes can be inhibited by disturbing heteromers of neutrophil HNP1 and platelet CCL5. Sci Transl Med. 2015;7(317):317ra196
Alard J-E, Ortega-Gomez A, Wichapong K, Bongiovanni D, Horckmans M, Megens RTA, Leoni G, Ferraro B, Rossaint J, Paulin N, Ng J, Ippel H, Suylen D, Hinkel R, Blanchet X, Gaillard F, D’Amico M, von Hundelshausen P, Zarbock A, Scheiermann C, Hackeng TM, Steffens S, Kupatt C, Nicolaes GAF, Weber C, Soehnlein O
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Cross-linking GPVI-Fc by anti-Fc antibodies potentiates its inhibition of atherosclerotic plaque- and collagen-induced platelet activation. JACC Basic Transl Sci. 2016 Apr 25;1(3):131-142
Jamasbi J, Megens RTA, Bianchini M, Uhland K, Münch G, Ungerer M, Sherman S, Faussner A, Brandl R, John C, Buchner J, Weber C, R Lorenz, Elia N, Siess W
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CD27 co-stimulation increases the abundance of regulatory T cells and reduces atherosclerosis in hyperlipidaemic mice. Eur Heart J. 2017 Dec 21;38(48):3590-3599
Winkels H, Meiler S, Lievens D, Engel D, Spitz C, Bürger C, Beckers L, Dandl A, Reim S, Ahmadsei M, Hartwig H, Holdt LM, Hristov M, Megens RTA, Schmitt MM, Biessen EA, Borst J, Faussner A, Weber C, Lutgens E, Gerdes N
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CD70 limits atherosclerosis and promotes macrophage function. Thromb Haemost. 2017;117(1):164-175
Winkels H, Meiler S, Smeets E, Lievens D, Engel D, Spitz C, Bürger C, Rinne P, Beckers L, Dandl A, Reim S, Ahmadsei M, Van den Bossche J, Holdt LM, Megens RTA, Schmitt MM, de Winther M, Biessen EA, Borst J, Faussner A, Weber C, Lutgens E, Gerdes N
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Chemokine interactome mapping enables tailored intervention in acute and chronic inflammation. Sci Transl Med. 2017 Apr 5;9(384)
von Hundelshausen P, Agten S, Eckardt V, Blanchet X, Schmitt MM, Ippel H, Neideck C, Bidzhekov K, Leberzammer J, Wichapong K, Faussner A, Drechsler M, Grommes J, van Geffen J, Li H, Ortega A, Megens RTA, Naumann R, Dijkgraaf I, Nicolaes GAF, Döring Y, Soehnlein O, Lutgens E, Heemskerk JWM, Koenen RR, Mayo KH, Hackeng TM, Weber C
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Chronic Intake of the Selective Serotonin Reuptake Inhibitor Fluoxetine Enhances Atherosclerosis. Arterioscler Thromb Vasc Biol. 2018;38(5):1007-19 PMID: 29567680
Rami, M, Guillamat-Prats R, Rinne P, Ring L, Söhnlein O, Blanchet X, Megens, RTA, Döring Y, Weber C, Faussner A, Steffens S
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Dimeric Glycoprotein VI Binds to Collagen but Not to Fibrin. Thromb Haemost 2018;118:351–361
Ebrahim M, Jamasbi J, Adler K, Megens RTA, M'Bengue Y, Blanchet X, Uhland K, Ungerer M, Brandl R, Weber C, Elia N, Lorenz R, Münch G, Siess W
