Objective: Fluid-phase pinocytosis is a receptor-independent mechanism of endocytosis that occurs in all mammalian cells and may be a mechanism for the uptake of LDL by macrophages. As there are currently no methods for the measurement of fluid-phase pinocytosis by individual aortic cells in vivo, we sought to identify a suitable method. Methods: ApoE-/- mice were retro-orbitally injected with AngioSPARK fluorescent nanoparticles specifically designed to not interact with cells. After 24 h, mice were sacrificed, and the aortas were isolated and then digested to analyze aortic cell uptake of AngioSPARK by flow cytometry. Results: CD11b-expressing aortic macrophages from mice injected with AngioSPARK showed high levels of fluid-phase pinocytosis compared to aortic cells not expressing CD11b (4,393.7 vs. 408.3 mean fluorescence intensity [MFI], respectively). Conclusion: This new technique allows for the measurement of fluid-phase pinocytosis by aortic cells in vivo, making it possible to examine the cell-signaling molecules and drugs that affect this process. Published by S. Karger AG, Basel

Keywords:
Fluid-phase pinocytosis, Macropinocytosis, Micropinocytosis, Macrophage, Flow cytometry, Atherosclerosis, Aorta
1.
Swanson JA, Watts C: Macropinocytosis. Trends Cell Biol 1995;5:424-428.
2.
Norbury CC: Drinking a lot is good for dendritic cells. Immunology 2006;117:443-451.
3.
Kerr MC, Teasdale RD: Defining macropinocytosis. Traffic 2009;10:364-371.
4.
Kruth HS, Jones NL, Huang W, Zhao B, Ishii I, Chang J, Combs CA, Malide D, Zhang WY: Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein. J Biol Chem 2005;280:2352-2360.
5.
Zhao B, Li Y, Buono C, Waldo SW, Jones NL, Mori M, Kruth HS: Constitutive receptor-independent low density lipoprotein uptake and cholesterol accumulation by macrophages differentiated from human monocytes with macrophage-colony-stimulating factor (M-CSF). J Biol Chem 2006;281:15757-15762.
6.
Buono C, Anzinger JJ, Amar M, Kruth HS: Fluorescent pegylated nanoparticles demonstrate fluid-phase pinocytosis by macrophages in mouse atherosclerotic lesions. J Clin Invest 2009;119:1373-1381.
7.
Anzinger JJ, Chang J, Xu Q, Barthwal MK, Bohnacker T, Wymann MP, Kruth HS: Murine bone marrow-derived macrophages differentiated with GM-CSF become foam cells by PI3Kγ-dependent fluid-phase pinocytosis of native LDL. J Lipid Res 2012;53:34-42.
8.
Anzinger JJ, Chang J, Xu Q, Buono C, Li Y, Leyva FJ, Park BC, Greene LE, Kruth HS: Native low-density lipoprotein uptake by macrophage colony-stimulating factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis. Arterioscler Thromb Vasc Biol 2010;30:2022-2031.
9.
Barthwal MK, Anzinger JJ, Xu Q, Bohnacker T, Wymann MP, Kruth HS: Fluid-phase pinocytosis of native low density lipoprotein promotes murine M-CSF-differentiated macrophage foam cell formation. PLoS One 2013;8:e58054.
10.
Sallusto F, Cella M, Danieli C, Lanzavecchia A: Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J Exp Med 1995;182:389-400.
11.
Kruth HS: Fluid-phase pinocytosis of LDL by macrophages: a novel target to reduce macrophage cholesterol accumulation in atherosclerotic lesions. Curr Pharm Des 2013;19:5865-5872.
12.
Groves K, Kossodo S, Naranayan N, Peterson J, Rajopadhye M: A new platform of superbright fluorescent nanoparticles for in vivo imaging. Paper, 5th Annu Meet Soc Molecular Imaging, Waikoloa, August 2006.
13.
Ishibashi S, Goldstein JL, Brown MS, Herz J, Burns DK: Massive xanthomatosis and atherosclerosis in cholesterol-fed low density lipoprotein receptor-negative mice. J Clin Invest 1994;93:1885-1893.
14.
Galkina E, Kadl A, Sanders J, Varughese D, Sarembock IJ, Ley K: Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent. J Exp Med 2006;203:1273-1282.
15.
Iuliano L, Mauriello A, Sbarigia E, Spagnoli LG, Violi F: Radiolabeled native low-density lipoprotein injected into patients with carotid stenosis accumulates in macrophages of atherosclerotic plaque : effect of vitamin E supplementation. Circulation 2000;101:1249-1254.
16.
Springer T, Galfre G, Secher DS, Milstein C: Mac-1: a macrophage differentiation antigen identified by monoclonal antibody. Eur J Immunol 1979;9:301-306.
17.
Cybulsky MI, Cheong C, Robbins CS: Macrophages and dendritic cells: partners in atherogenesis. Circ Res 2016;118:637-652.
18.
Koltsova EK, Hedrick CC, Ley K: Myeloid cells in atherosclerosis: a delicate balance of anti-inflammatory and proinflammatory mechanisms. Curr Opin Lipidol 2013;24:371-380.
19.
Koltsova EK, Garcia Z, Chodaczek G, Landau M, McArdle S, Scott SR, von Vietinghoff S, Galkina E, Miller YI, Acton ST, Ley K: Dynamic T cell-APC interactions sustain chronic inflammation in atherosclerosis. J Clin Invest 2012;122:3114-3126.
20.
Swanson JA: Shaping cups into phagosomes and macropinosomes. Nat Rev Mol Cell Biol 2008;9:639-649.
21.
Lim JP, Gleeson PA: Macropinocytosis: an endocytic pathway for internalising large gulps. Immunol Cell Biol 2011;89:836-843.
22.
Armstrong JK, Wenby RB, Meiselman HJ, Fisher TC: The hydrodynamic radii of macromolecules and their effect on red blood cell aggregation. Biophys J 2004;87:4259-4270.
23.
Vasile E, Simionescu M, Simionescu N: Visualization of the binding, endocytosis, and transcytosis of low-density lipoprotein in the arterial endothelium in situ. J Cell Biol 1983;96:1677-1689.
24.
Steinberg D, Pittman RC, Carew TE: Mechanisms involved in the uptake and degradation of low density lipoprotein by the artery wall in vivo. Ann NY Acad Sci 1985;454:195-206.
25.
Kruth HS: Receptor-independent fluid-phase pinocytosis mechanisms for induction of foam cell formation with native low-density lipoprotein particles. Curr Opin Lipidol 2011;22:386-393.
26.
Cossart P, Helenius A: Endocytosis of viruses and bacteria. Cold Spring Harb Perspect Biol 2014;6:a016972.
27.
Ha KD, Bidlingmaier SM, Liu B: Macropinocytosis exploitation by cancers and cancer therapeutics. Front Physiol 2016;7:381.
Published by S. Karger AG, Basel
2017
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.