Abstract
Recruitment of leukocytes into arteries is a hallmark event throughout all stages of atherosclerosis and hence stands out as a primary therapeutic target. To understand the molecular mechanisms of arterial leukocyte subset infiltration, real-time visualization of recruitment processes of leukocyte subsets at high resolution is a prerequisite. In this review we provide a balanced overview of optical imaging modalities in the more commonly used experimental models for atherosclerosis (e.g., mouse models) allowing for in vivo display of recruitment processes in large arteries and further detail strategies to overcome hurdles inherent to arterial imaging. We further provide a synopsis of techniques allowing for non-toxic, photostable labeling of target structures. Finally, we deliver a short summary of ongoing developments including the emergence of novel labeling approaches, the use of superresolution microscopy, and the potentials of opto-acoustic microscopy and intravascular 2-dimensional near-infrared fluorescence microscopy.
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References
Aulchenko YS, Ripatti S, Lindqvist I et al (2009) Loci influencing lipid levels and coronary heart disease risk in 16 European population cohorts. Nat Genet 41:47–55
Willer CJ, Sanna S, Jackson AU et al (2008) Newly identified loci that influence lipid concentrations and risk of coronary artery disease. Nat Genet 40:161–169
Soehnlein O, Drechsler M, Hristov M et al (2009) Functional alterations of myeloid cell subsets in hyperlipidaemia: relevance for atherosclerosis. J Cell Mol Med 13:4293–4303
Weber C, Noels H (2011) Atherosclerosis: current pathogenesis and therapeutic options. Nat Med 17:1410–1422
Drechsler M, Megens RT, van Zandvoort M et al (2010) Hyperlipidemia-triggered neutrophilia promotes early atherosclerosis. Circulation 122:1837–1845
Soehnlein O, Drechsler M, Doring Y et al (2013) Distinct functions of chemokine receptor axes in the atherogenic mobilization and recruitment of classical monocytes. EMBO Mol Med 5:471–481
Wantha S, Alard JE, Megens RT et al (2013) Neutrophil-derived cathelicidin promotes adhesion of classical monocytes. Circ Res 112:792–801
Doring Y, Drechsler M, Wantha S et al (2012) Lack of neutrophil-derived cramp reduces atherosclerosis in mice. Circ Res 110:1052–1056
Soehnlein O, Xie X, Ulbrich H et al (2005) Neutrophil-derived heparin-binding protein (hbp/cap37) deposited on endothelium enhances monocyte arrest under flow conditions. J Immunol 174:6399–6405
Llodra J, Angeli V, Liu J et al (2004) Emigration of monocyte-derived cells from atherosclerotic lesions characterizes regressive, but not progressive, plaques. Proc Natl Acad Sci U S A 101:11779–11784
Robbins CS, Hilgendorf I, Weber GF et al (2013) Local proliferation dominates lesional macrophage accumulation in atherosclerosis. Nat Med 19:1166–1172
Eriksson EE (2011) Intravital microscopy on atherosclerosis in apolipoprotein e-deficient mice establishes microvessels as major entry pathways for leukocytes to advanced lesions. Circulation 124:2129–2138
Hellings WE, Peeters W, Moll FL et al (2010) Composition of carotid atherosclerotic plaque is associated with cardiovascular outcome: a prognostic study. Circulation 121:1941–1950
Sluimer JC, Kolodgie FD, Bijnens AP et al (2009) Thin-walled microvessels in human coronary atherosclerotic plaques show incomplete endothelial junctions relevance of compromised structural integrity for intraplaque microvascular leakage. J Am Coll Cardiol 53:1517–1527
Silvestre-Roig C, de Winther MP, Weber C et al (2014) Atherosclerotic plaque destabilization: mechanisms, models, and therapeutic strategies. Circ Res 114:214–226
Lee S, Vinegoni C, Feruglio PF et al (2012) Real-time in vivo imaging of the beating mouse heart at microscopic resolution. Nat Commun 3:1054
Lammermann T, Afonso PV, Angermann BR et al (2013) Neutrophil swarms require ltb4 and integrins at sites of cell death in vivo. Nature 498:371–375
Looney MR, Thornton EE, Sen D et al (2011) Stabilized imaging of immune surveillance in the mouse lung. Nat Methods 8:91–96
Leuschner F, Panizzi P, Chico-Calero I et al (2010) Angiotensin-converting enzyme inhibition prevents the release of monocytes from their splenic reservoir in mice with myocardial infarction. Circ Res 107:1364–1373
van Gestel MA, Heemskerk JW, Slaaf DW et al (2003) In vivo blockade of platelet adp receptor p2y12 reduces embolus and thrombus formation but not thrombus stability. Arterioscler Thromb Vasc Biol 23:518–523
Proebstl D, Voisin MB, Woodfin A et al (2012) Pericytes support neutrophil subendothelial cell crawling and breaching of venular walls in vivo. J Exp Med 209:1219–1234
Soehnlein O, Zernecke A, Eriksson EE et al (2008) Neutrophil secretion products pave the way for inflammatory monocytes. Blood 112:1461–1471
Kempf T, Zarbock A, Widera C et al (2011) Gdf-15 is an inhibitor of leukocyte integrin activation required for survival after myocardial infarction in mice. Nat Med 17:581–588
Megens RT, Reitsma S, Prinzen L et al (2010) In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy. J Biomed Opt 15:011108
Reitsma S, Oude Egbrink MG, Heijnen VV et al (2011) Endothelial glycocalyx thickness and platelet-vessel wall interactions during atherogenesis. Thromb Haemost 106:939–946
Soehnlein O, Wantha S, Simsekyilmaz S et al (2011) Neutrophil-derived cathelicidin protects from neointimal hyperplasia. Sci Transl Med 3:103ra198
Lutgens E, Lievens D, Beckers L et al (2010) Deficient cd40-traf6 signaling in leukocytes prevents atherosclerosis by skewing the immune response toward an antiinflammatory profile. J Exp Med 207:391–404
Massberg S, Gawaz M, Gruner S et al (2003) A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo. J Exp Med 197:41–49
Yipp BG, Petri B, Salina D et al (2012) Infection-induced netosis is a dynamic process involving neutrophil multitasking in vivo. Nat Med 18:1386–1393
Wilson T (2010) Spinning-disk microscopy systems. Cold Spring Harb Protoc 2010:pdb.top88
Fan GY, Fujisaki H, Miyawaki A et al (1999) Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons. Biophys J 76:2412–2420
Chevre R, Gonzalez-Granado JM, Megens RT et al (2014) High-resolution imaging of intravascular atherogenic inflammation in live mice. Circ Res 114:770–779
van Zandvoort M, Engels W, Douma K et al (2004) Two-photon microscopy for imaging of the (atherosclerotic) vascular wall: a proof of concept study. J Vasc Res 41:54–63
Niesner RA, Andresen V, Gunzer M (2008) Intravital two-photon microscopy: focus on speed and time resolved imaging modalities. Immunol Rev 221:7–25
Kurtz R, Fricke M, Kalb J et al (2006) Application of multiline two-photon microscopy to functional in vivo imaging. J Neurosci Methods 151:276–286
Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248:73–76
Janssen BJ, De Celle T, Debets JJ et al (2004) Effects of anesthetics on systemic hemodynamics in mice. Am J Physiol Heart Circ Physiol 287:H1618–H1624
Kreisel D, Nava RG, Li W et al (2010) In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation. Proc Natl Acad Sci U S A 107:18073–18078
Lockett AD, Brown MB, Santos-Falcon N et al (2014) Active trafficking of alpha 1 antitrypsin across the lung endothelium. PLoS One 9:e93979
Rademakers T, Douma K, Hackeng TM et al (2013) Plaque-associated vasa vasorum in aged apolipoprotein e-deficient mice exhibit proatherogenic functional features in vivo. Arterioscler Thromb Vasc Biol 33:249–256
Schmitt MM, Megens RT, Zernecke A et al (2014) Endothelial junctional adhesion molecule-a guides monocytes into flow-dependent predilection sites of atherosclerosis. Circulation 129:66–76
Megens RT, Vijayan S, Lievens D et al (2012) Presence of luminal neutrophil extracellular traps in atherosclerosis. Thromb Haemost 107:597–598
Reutelingsperger CP, Dumont E, Thimister PW et al (2002) Visualization of cell death in vivo with the annexin a5 imaging protocol. J Immunol Methods 265:123–132
Yu W, Braz JC, Dutton AM et al (2007) In vivo imaging of atherosclerotic plaques in apolipoprotein e deficient mice using nonlinear microscopy. J Biomed Opt 12:054008
Nahrendorf M, Swirski FK (2014) Fluorescent leukocytes enter plaque on the microscope stage. Circ Res 114:740–741
Jung S, Aliberti J, Graemmel P et al (2000) Analysis of fractalkine receptor cx(3)cr1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol 20:4106–4114
Tacke F, Alvarez D, Kaplan TJ et al (2007) Monocyte subsets differentially employ ccr2, ccr5, and cx3cr1 to accumulate within atherosclerotic plaques. J Clin Invest 117:185–194
Saederup N, Cardona AE, Croft K et al (2010) Selective chemokine receptor usage by central nervous system myeloid cells in ccr2-red fluorescent protein knock-in mice. PLoS One 5:e13693
Abe T, Sakaue-Sawano A, Kiyonari H et al (2013) Visualization of cell cycle in mouse embryos with fucci2 reporter directed by rosa26 promoter. Development 140:237–246
Livet J, Weissman TA, Kang H et al (2007) Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450:56–62
Stockholm D, Bartoli M, Sillon G et al (2005) Imaging calpain protease activity by multiphoton fret in living mice. J Mol Biol 346:215–222
Kardash E, Reichman-Fried M, Maitre JL et al (2010) A role for rho gtpases and cell-cell adhesion in single-cell motility in vivo. Nat Cell Biol 12:47–53, sup pp 1–11
Kedrin D, Gligorijevic B, Wyckoff J et al (2008) Intravital imaging of metastatic behavior through a mammary imaging window. Nat Methods 5:1019–1021
Victora GD, Schwickert TA, Fooksman DR et al (2010) Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell 143:592–605
Last’ovicka J, Budinsky V, Spisek R et al (2009) Assessment of lymphocyte proliferation: Cfse kills dividing cells and modulates expression of activation markers. Cell Immunol 256:79–85
Parish CR (1999) Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol Cell Biol 77:499–508
Zhao H, Traganos F, Dobrucki J et al (2009) Induction of DNA damage response by the supravital probes of nucleic acids. Cytometry A 75:510–519
Mempel TR, Pittet MJ, Khazaie K et al (2006) Regulatory t cells reversibly suppress cytotoxic t cell function independent of effector differentiation. Immunity 25:129–141
Horan PK, Slezak SE (1989) Stable cell membrane labelling. Nature 340:167–168
Lo Celso C, Fleming HE, Wu JW et al (2009) Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature 457:92–96
Joseph C, Quach JM, Walkley CR et al (2013) Deciphering hematopoietic stem cells in their niches: a critical appraisal of genetic models, lineage tracing, and imaging strategies. Cell Stem Cell 13:520–533
Chiang EY, Hidalgo A, Chang J et al (2007) Imaging receptor microdomains on leukocyte subsets in live mice. Nat Methods 4:219–222
Daley JM, Thomay AA, Connolly MD et al (2008) Use of ly6g-specific monoclonal antibody to deplete neutrophils in mice. J Leukoc Biol 83:64–70
Wang JX, Bair AM, King SL et al (2012) Ly6g ligation blocks recruitment of neutrophils via a beta2-integrin-dependent mechanism. Blood 120:1489–1498
Kunkel EJ, Chomas JE, Ley K (1998) Role of primary and secondary capture for leukocyte accumulation in vivo. Circ Res 82:30–38
Moreau HD, Lemaitre F, Terriac E et al (2012) Dynamic in situ cytometry uncovers t cell receptor signaling during immunological synapses and kinapses in vivo. Immunity 37:351–363
Zhao W, Schafer S, Choi J et al (2011) Cell-surface sensors for real-time probing of cellular environments. Nat Nanotechnol 6:524–531
Shcherbo D, Shemiakina II, Ryabova AV et al (2010) Near-infrared fluorescent proteins. Nat Methods 7:827–829
Shu X, Royant A, Lin MZ et al (2009) Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome. Science 324:804–807
Filonov GS, Piatkevich KD, Ting LM et al (2011) Bright and stable near-infrared fluorescent protein for in vivo imaging. Nat Biotechnol 29:757–761
Herz J, Siffrin V, Hauser AE et al (2010) Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator. Biophys J 98:715–723
Kawano H, Kogure T, Abe Y et al (2008) Two-photon dual-color imaging using fluorescent proteins. Nat Methods 5:373–374
Abbe E (1873) Ueber einen neuen beleuchtungsapparat am mikroskop. In: Schultze M (ed) Archive für mikrosckopische anatomie. Verlag von Max Cohen Sohn, Bonn, pp 469–481
Hell SW (2007) Far-field optical nanoscopy. Science 316:1153–1158
Muller T, Schumann C, Kraegeloh A (2012) Sted microscopy and its applications: new insights into cellular processes on the nanoscale. ChemPhysChem 13:1986–2000
Lauterbach MA, Keller J, Schonle A et al (2010) Comparing video-rate sted nanoscopy and confocal microscopy of living neurons. J Biophotonics 3:417–424
Berning S, Willig KI, Steffens H et al (2012) Nanoscopy in a living mouse brain. Science 335:551
Wurm CA, Kolmakov K, Göttfert F et al (2012) Novel red fluorophores with superior performance in sted microscopy. Opt Nanoscopy 1:1–7
Lavoie-Cardinal F, Jensen NA, Westphal V et al (2014) Two-color resolft nanoscopy with green and red fluorescent photochromic proteins. ChemPhysChem 15:655–663
Grotjohann T, Testa I, Leutenegger M et al (2011) Diffraction-unlimited all-optical imaging and writing with a photochromic gfp. Nature 478:204–208
Grotjohann T, Testa I, Reuss M et al (2012) Rsegfp2 enables fast resolft nanoscopy of living cells. eLife 1:e00248
Chmyrov A, Keller J, Grotjohann T et al (2013) Nanoscopy with more than 100,000 ‘doughnuts’. Nat Methods 10:737–740
Nienhaus K, Nienhaus GU (2014) Fluorescent proteins for live-cell imaging with super-resolution. Chem Soc Rev 43:1088–1106
Ntziachristos V, Razansky D (2010) Molecular imaging by means of multispectral optoacoustic tomography (msot). Chem Rev 110:2783–2794
Buehler A, Herzog E, Razansky D et al (2010) Video rate optoacoustic tomography of mouse kidney perfusion. Opt Lett 35:2475–2477
Ma R, Taruttis A, Ntziachristos V et al (2009) Multispectral optoacoustic tomography (msot) scanner for whole-body small animal imaging. Opt Express 17:21414–21426
Razansky D, Vinegoni C, Ntziachristos V (2009) Imaging of mesoscopic-scale organisms using selective-plane optoacoustic tomography. Phys Med Biol 54:2769–2777
Razansky D, Deliolanis NC, Vinegoni C et al (2012) Deep tissue optical and optoacoustic molecular imaging technologies for pre-clinical research and drug discovery. Curr Pharm Biotechnol 13:504–522
Omar M, Gateau J, Ntziachristos V (2013) Raster-scan optoacoustic mesoscopy in the 25-125 mhz range. Opt Lett 38:2472–2474
Tserevelakis GJ, Soliman D, Omar M et al (2014) Hybrid multiphoton and optoacoustic microscope. Opt Lett 39:1819–1822
Taruttis A, Ntziachristos V (2012) Translational optical imaging. AJR Am J Roentgenol 199:263–271
Ntziachristos V, Razansky D (2013) Optical and opto-acoustic imaging. Recent Results Cancer Res 187:133–150
Yao J, Wang LV (2013) Photoacoustic microscopy. Laser Photon Rev 7:758
Jaffer FA, Calfon MA, Rosenthal A et al (2011) Two-dimensional intravascular near-infrared fluorescence molecular imaging of inflammation in atherosclerosis and stent-induced vascular injury. J Am Coll Cardiol 57:2516–2526
Calfon MA, Rosenthal A, Mallas G, et al (2011) In vivo near infrared fluorescence (nirf) intravascular molecular imaging of inflammatory plaque, a multimodal approach to imaging of atherosclerosis. J Vis Exp (54):e2257
Sasmono RT, Oceandy D, Pollard JW et al (2003) A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse. Blood 101:1155–1163
Faust N, Varas F, Kelly LM et al (2000) Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood 96:719–726
Boes M, Cerny J, Massol R et al (2002) T-cell engagement of dendritic cells rapidly rearranges mhc class ii transport. Nature 418:983–988
Acknowledgements
The work of the authors’ is supported by the DFG (SO876/3-1, SO876/6-1, SFB914 TP B08, SFB1123 TPs A06, B05, Z01, INST409/97-1), the Else Kröner Fresenius Stiftung, the NWO (VIDI project 91712303), the LMUexcellence, and the FöFoLe program of the LMU Munich.
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None declared.
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Megens, R.T.A., Soehnlein, O. (2015). Intravital Microscopy for Atherosclerosis Research. In: Andrés, V., Dorado, B. (eds) Methods in Mouse Atherosclerosis. Methods in Molecular Biology, vol 1339. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2929-0_3
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