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Non-invasive dynamic near-infrared imaging and quantification of vascular leakage in vivo

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Abstract

Preclinical vascular research has been hindered by a lack of methods that can sensitively image and quantify vascular perfusion and leakage in vivo. In this study, we have developed dynamic near-infrared imaging methods to repeatedly visualize and quantify vascular leakage in mouse skin in vivo, and we have applied these methods to transgenic mice with overexpression of vascular endothelial growth factors VEGF-A or -C. Near-infrared dye conjugates were developed to identify a suitable vascular tracer that had a prolonged circulation lifetime and slow leakage into normal tissue after intravenous injection. Dynamic simultaneous imaging of ear skin and a large blood vessel in the leg enabled determination of the intravascular signal (blood volume fraction) from the tissue signal shortly after injection and quantifications of vascular leakage into the extravascular tissue over time. This method allowed for the sensitive detection of increased blood vascularity and leakage rates in K14-VEGF-A transgenic mice and also reliably measured inflammation-induced changes of vascularity and leakage over time in the same mice. Measurements after injection of recombinant VEGF-A surprisingly revealed increased blood vascular leakage and lymphatic clearance in K14-VEGF-C transgenic mice which have an expanded cutaneous lymphatic vessel network, potentially indicating unanticipated effects of lymphatic drainage on vascular leakage. Increased vascular leakage was also detected in subcutaneous tumors, confirming that the method can also be applied to deeper tissues. This new imaging method might facilitate longitudinal investigations of the in vivo effects of drug candidates, including angiogenesis inhibitors, in preclinical disease models.

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References

  1. Yuan SY, Rigor RR (2010) In: Regulation of Endothelial Barrier Function. Integrated Systems Physiology: From Molecule to Function to Disease. San Rafael,CA

  2. Levick JR, Michel CC (2010) Microvascular fluid exchange and the revised Starling principle. Cardiovasc Res 87(2):198–210. doi:10.1093/cvr/cvq062

    Article  PubMed  CAS  Google Scholar 

  3. Scallan J, Huxley VH, Korthuis RJ (2010). In: Capillary fluid exchange: regulation, functions, and pathology. Integrated systems physiology: from molecule to function to disease. San Rafael, CA

  4. Nagy JA, Dvorak AM, Dvorak HF (2007) VEGF-A and the induction of pathological angiogenesis. Annu Rev Pathol 2:251–275. doi:10.1146/annurev.pathol.2.010506.134925

    Article  PubMed  CAS  Google Scholar 

  5. Dejana E, Tournier-Lasserve E, Weinstein BM (2009) The control of vascular integrity by endothelial cell junctions: molecular basis and pathological implications. Dev Cell 16(2):209–221. doi:10.1016/j.devcel.2009.01.004

    Article  PubMed  CAS  Google Scholar 

  6. Miles AA, Miles EM (1952) Vascular reactions to histamine, histamine-liberator and leukotaxine in the skin of guinea-pigs. J Physiol 118(2):228–257

    PubMed  CAS  Google Scholar 

  7. Nagy JA, Benjamin L, Zeng H, Dvorak AM, Dvorak HF (2008) Vascular permeability, vascular hyperpermeability and angiogenesis. Angiogenesis 11(2):109–119. doi:10.1007/s10456-008-9099-z

    Article  PubMed  CAS  Google Scholar 

  8. Bates DO (2010) Vascular endothelial growth factors and vascular permeability. Cardiovasc Res 87(2):262–271. doi:10.1093/cvr/cvq105

    Article  PubMed  CAS  Google Scholar 

  9. Curry FR, Adamson RH (2010) Vascular permeability modulation at the cell, microvessel, or whole organ level: towards closing gaps in our knowledge. Cardiovasc Res 87(2):218–229. doi:10.1093/cvr/cvq115

    Article  PubMed  CAS  Google Scholar 

  10. Kenne E, Lindbom L (2011) Imaging inflammatory plasma leakage in vivo. Thromb Haemost 105(5):783–789. doi:10.1160/TH10-10-0635

    Article  PubMed  CAS  Google Scholar 

  11. Neeman M, Dafni H (2003) Structural, functional, and molecular MR imaging of the microvasculature. Annu Rev Biomed Eng 5:29–56. doi:10.1146/annurev.bioeng.5.040202.121606

    Article  PubMed  CAS  Google Scholar 

  12. Mulder WJ, Griffioen AW (2010) Imaging of angiogenesis. Angiogenesis 13(2):71–74. doi:10.1007/s10456-010-9178-9

    Article  PubMed  Google Scholar 

  13. Dreher MR, Liu W, Michelich CR, Dewhirst MW, Yuan F, Chilkoti A (2006) Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers. J Natl Cancer Inst 98(5):335–344. doi:10.1093/jnci/djj070

    Article  PubMed  CAS  Google Scholar 

  14. Vandoorne K, Addadi Y, Neeman M (2010) Visualizing vascular permeability and lymphatic drainage using labeled serum albumin. Angiogenesis 13(2):75–85. doi:10.1007/s10456-010-9170-4

    Article  PubMed  CAS  Google Scholar 

  15. Hilderbrand SA, Weissleder R (2010) Near-infrared fluorescence: application to in vivo molecular imaging. Curr Opin Chem Biol 14(1):71–79. doi:10.1016/j.cbpa.2009.09.029

    Article  PubMed  CAS  Google Scholar 

  16. Cherrick GR, Stein SW, Leevy CM, Davidson CS (1960) Indocyanine green: observations on its physical properties, plasma decay, and hepatic extraction. J Clin Invest 39:592–600. doi:10.1172/JCI104072

    Article  PubMed  CAS  Google Scholar 

  17. Jeltsch M, Kaipainen A, Joukov V, Meng X, Lakso M, Rauvala H, Swartz M, Fukumura D, Jain RK, Alitalo K (1997) Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science 276(5317):1423–1425

    Article  PubMed  CAS  Google Scholar 

  18. Xia YP, Li B, Hylton D, Detmar M, Yancopoulos GD, Rudge JS (2003) Transgenic delivery of VEGF to mouse skin leads to an inflammatory condition resembling human psoriasis. Blood 102(1):161–168. doi:10.1182/blood-2002-12-3793

    Article  PubMed  CAS  Google Scholar 

  19. Thurston G, Suri C, Smith K, McClain J, Sato TN, Yancopoulos GD, McDonald DM (1999) Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. Science 286(5449):2511–2514

    Article  PubMed  CAS  Google Scholar 

  20. Wyatt SK, Manning HC, Bai M, Bailey SN, Gallant P, Ma G, McIntosh L, Bornhop DJ (2010) Molecular imaging of the translocator protein (TSPO) in a pre-clinical model of breast cancer. Mol Imaging Biol 12(3):349–358. doi:10.1007/s11307-009-0270-8

    Article  PubMed  Google Scholar 

  21. Ott P, Keiding S, Bass L (1993) Plasma elimination of indocyanine green in the intact pig after bolus injection and during constant infusion: comparison of spectrophotometry and high-pressure liquid chromatography for concentration analysis. Hepatology 18(6):1504–1515. doi:10.1002/hep.1840180633

    PubMed  CAS  Google Scholar 

  22. Kunstfeld R, Hirakawa S, Hong YK, Schacht V, Lange-Asschenfeldt B, Velasco P, Lin C, Fiebiger E, Wei X, Wu Y, Hicklin D, Bohlen P, Detmar M (2004) Induction of cutaneous delayed-type hypersensitivity reactions in VEGF-A transgenic mice results in chronic skin inflammation associated with persistent lymphatic hyperplasia. Blood 104(4):1048–1057. doi:10.1182/blood-2003-08-2964

    Article  PubMed  CAS  Google Scholar 

  23. Detmar M, Brown LF, Schon MP, Elicker BM, Velasco P, Richard L, Fukumura D, Monsky W, Claffey KP, Jain RK (1998) Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice. J Invest Dermatol 111(1):1–6. doi:10.1046/j.1523-1747.1998.00262.x

    Article  PubMed  CAS  Google Scholar 

  24. Halin C, Detmar M (2008) Chapter 1. Inflammation, angiogenesis, and lymphangiogenesis. Methods Enzymol 445:1–25. doi:10.1016/S0076-6879(08)03001-2

    Article  PubMed  CAS  Google Scholar 

  25. Dafni H, Landsman L, Schechter B, Kohen F, Neeman M (2002) MRI and fluorescence microscopy of the acute vascular response to VEGF165: vasodilation, hyper-permeability and lymphatic uptake, followed by rapid inactivation of the growth factor. NMR Biomed 15(2):120–131

    Article  PubMed  CAS  Google Scholar 

  26. Lohela M, Helotera H, Haiko P, Dumont DJ, Alitalo K (2008) Transgenic induction of vascular endothelial growth factor-C is strongly angiogenic in mouse embryos but leads to persistent lymphatic hyperplasia in adult tissues. Am J Pathol 173(6):1891–1901. doi:10.2353/ajpath.2008.080378

    Article  PubMed  CAS  Google Scholar 

  27. Huggenberger R, Siddiqui SS, Brander D, Ullmann S, Zimmermann K, Antsiferova M, Werner S, Alitalo K, Detmar M (2011) An important role of lymphatic vessel activation in limiting acute inflammation. Blood 117(17):4667–4678. doi:10.1182/blood-2010-10-316356

    Article  PubMed  CAS  Google Scholar 

  28. Krynyckyi BR, Kim CK, Goyenechea MR, Chan PT, Zhang ZY, Machac J (2004) Clinical breast lymphoscintigraphy: optimal techniques for performing studies, image atlas, and analysis of images. Radiographics 24 (1):121–145; discussion 139–145. doi:10.1148/rg.241025713

    Google Scholar 

  29. Karlsen TV, McCormack E, Mujic M, Tenstad O, Wiig H (2011) Minimally-invasive quantification of lymph flow in mice and rats by imaging depot clearance of near-infrared albumin. Am J Physiol Heart Circ Physiol. doi:10.1152/ajpheart.00842.2011

    PubMed  Google Scholar 

  30. Tammela T, Alitalo K (2010) Lymphangiogenesis: molecular mechanisms and future promise. Cell 140(4):460–476. doi:10.1016/j.cell.2010.01.045

    Article  PubMed  CAS  Google Scholar 

  31. Joukov V, Kumar V, Sorsa T, Arighi E, Weich H, Saksela O, Alitalo K (1998) A recombinant mutant vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding, activation, and vascular permeability activities. J Biol Chem 273(12):6599–6602

    Article  PubMed  CAS  Google Scholar 

  32. Saxena V, Sadoqi M, Shao J (2003) Degradation kinetics of indocyanine green in aqueous solution. J Pharm Sci 92(10):2090–2097. doi:10.1002/jps.10470

    Article  PubMed  CAS  Google Scholar 

  33. Troyan SL, Kianzad V, Gibbs-Strauss SL, Gioux S, Matsui A, Oketokoun R, Ngo L, Khamene A, Azar F, Frangioni JV (2009) The FLARE intraoperative near-infrared fluorescence imaging system: a first-in-human clinical trial in breast cancer sentinel lymph node mapping. Ann Surg Oncol 16(10):2943–2952. doi:10.1245/s10434-009-0594-2

    Article  PubMed  Google Scholar 

  34. Marshall MV, Draney D, Sevick-Muraca EM, Olive DM (2010) Single-dose intravenous toxicity study of IRDye 800CW in Sprague-Dawley rats. Mol Imaging Biol 12(6):583–594. doi:10.1007/s11307-010-0317-x

    Article  PubMed  Google Scholar 

  35. Sandanaraj BS, Gremlich HU, Kneuer R, Dawson J, Wacha S (2010) Fluorescent nanoprobes as a biomarker for increased vascular permeability: implications in diagnosis and treatment of cancer and inflammation. Bioconjug Chem 21(1):93–101. doi:10.1021/bc900311h

    Article  PubMed  CAS  Google Scholar 

  36. Proulx ST, Luciani P, Derzsi S, Rinderknecht M, Mumprecht V, Leroux JC, Detmar M (2010) Quantitative imaging of lymphatic function with liposomal indocyanine green. Cancer Res 70(18):7053–7062. doi:10.1158/0008-5472.CAN-10-0271

    Article  PubMed  CAS  Google Scholar 

  37. Greenwald RB, Choe YH, McGuire J, Conover CD (2003) Effective drug delivery by PEGylated drug conjugates. Adv Drug Deliv Rev 55(2):217–250

    Article  PubMed  CAS  Google Scholar 

  38. Fox ME, Szoka FC, Frechet JM (2009) Soluble polymer carriers for the treatment of cancer: the importance of molecular architecture. Acc Chem Res 42(8):1141–1151. doi:10.1021/ar900035f

    Article  PubMed  CAS  Google Scholar 

  39. Huggenberger R, Ullmann S, Proulx ST, Pytowski B, Alitalo K, Detmar M (2010) Stimulation of lymphangiogenesis via VEGFR-3 inhibits chronic skin inflammation. J Exp Med 207(10):2255–2269. doi:10.1084/jem.20100559

    Article  PubMed  CAS  Google Scholar 

  40. Zhou Q, Guo R, Wood R, Boyce BF, Liang Q, Wang YJ, Schwarz EM, Xing L (2011) Vascular endothelial growth factor C attenuates joint damage in chronic inflammatory arthritis by accelerating local lymphatic drainage in mice. Arthritis Rheum 63(8):2318–2328. doi:10.1002/art.30421

    Article  PubMed  CAS  Google Scholar 

  41. Saaristo A, Tammela T, Farkkila A, Karkkainen M, Suominen E, Yla-Herttuala S, Alitalo K (2006) Vascular endothelial growth factor-C accelerates diabetic wound healing. Am J Pathol 169(3):1080–1087. doi:10.2353/ajpath.2006.051251

    Article  PubMed  CAS  Google Scholar 

  42. Baluk P, Tammela T, Ator E, Lyubynska N, Achen MG, Hicklin DJ, Jeltsch M, Petrova TV, Pytowski B, Stacker SA, Yla-Herttuala S, Jackson DG, Alitalo K, McDonald DM (2005) Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation. J Clin Invest 115(2):247–257. doi:10.1172/JCI22037

    PubMed  CAS  Google Scholar 

  43. Proulx ST, Kwok E, You Z, Beck CA, Shealy DJ, Ritchlin CT, Boyce BF, Xing L, Schwarz EM (2007) MRI and quantification of draining lymph node function in inflammatory arthritis. Ann N Y Acad Sci 1117:106–123. doi:10.1196/annals.1402.016

    Article  PubMed  Google Scholar 

  44. Greish K (2007) Enhanced permeability and retention of macromolecular drugs in solid tumors: a royal gate for targeted anticancer nanomedicines. J Drug Target 15(7–8):457–464. doi:10.1080/10611860701539584

    Article  PubMed  CAS  Google Scholar 

  45. Jain RK, Stylianopoulos T (2010) Delivering nanomedicine to solid tumors. Nat Rev Clin Oncol 7(11):653–664. doi:10.1038/nrclinonc.2010.139

    Article  PubMed  CAS  Google Scholar 

  46. Witzenbichler B, Asahara T, Murohara T, Silver M, Spyridopoulos I, Magner M, Principe N, Kearney M, Hu JS, Isner JM (1998) Vascular endothelial growth factor-C (VEGF-C/VEGF-2) promotes angiogenesis in the setting of tissue ischemia. Am J Pathol 153(2):381–394. doi:10.1016/S0002-9440(10)65582-4

    Article  PubMed  CAS  Google Scholar 

  47. Saaristo A, Veikkola T, Enholm B, Hytonen M, Arola J, Pajusola K, Turunen P, Jeltsch M, Karkkainen MJ, Kerjaschki D, Bueler H, Yla-Herttuala S, Alitalo K (2002) Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes. FASEB J 16(9):1041–1049. doi:10.1096/fj.01-1042com

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors would like to thank Carlos Ochoa for assistance with animal care and Sinem Karaman and Alexandra Ochsenbein for technical assistance. This work was supported by a Whitaker International Scholar grant (to S.T.P.); National Institutes of Health grant CA69184, Swiss National Science Foundation grants 3100A0-108207 and 31003A-130627, Commission of the European Communities grant LSHC-CT-2005-518178, Advanced European Research Council grant LYVICAM, Oncosuisse and Krebsliga Zurich (to M.D.).

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, ETH Zurich, Wolfgang-Pauli-Str. 10, HCI H303, 8093, Zurich, Switzerland

    Steven T. Proulx, Annamari Alitalo, Viviane Mumprecht, Ailsa J. Christiansen, Reto Huggenberger & Michael Detmar

  2. Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, ETH Zurich, Wolfgang-Pauli-Str. 10, HCI H301, 8093, Zurich, Switzerland

    Paola Luciani & Jean-Christophe Leroux

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  1. Steven T. Proulx
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  2. Paola Luciani
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  3. Annamari Alitalo
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  4. Viviane Mumprecht
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  5. Ailsa J. Christiansen
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  7. Jean-Christophe Leroux
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Correspondence to Jean-Christophe Leroux or Michael Detmar.

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Steven T. Proulx and Paola Luciani contributed equally to this work.

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Proulx, S.T., Luciani, P., Alitalo, A. et al. Non-invasive dynamic near-infrared imaging and quantification of vascular leakage in vivo. Angiogenesis 16, 525–540 (2013). https://doi.org/10.1007/s10456-013-9332-2

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