April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Pro-Vascular Mechanisms and in vivo Imaging Strategies of CCR3, a Specific Biomarker for Neovascular AMD
Author Affiliations & Notes
  • M. E. Kleinman
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky
  • W. Cho
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky
  • J. Z. Baffi
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky
  • R. J. C. Albuquerque
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky
  • M. Nozaki
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky
  • H. Kaneko
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky
  • K. Saito
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky
  • M. G. Rich
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky
  • M. E. Rothenberg
    Division of Allergy and Immunology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
  • J. Ambati
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky
  • Footnotes
    Commercial Relationships  M.E. Kleinman, University of Kentucky, P; W. Cho, None; J.Z. Baffi, None; R.J.C. Albuquerque, None; M. Nozaki, None; H. Kaneko, None; K. Saito, None; M.G. Rich, None; M.E. Rothenberg, None; J. Ambati, Allergan, C; Quark Pharmaceuticals, C; Novartis, C; University of Kentucky, P.
  • Footnotes
    Support  IRRF, NEI/NIH, Doris Duke Distinguished Clinical Scientist Award, Burroughs Wellcome Fund Clinical Scientist Award in Translational Research, Dr. E. Vernon Smith and Eloise C. Smith
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 4303. doi:
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    • Get Citation

      M. E. Kleinman, W. Cho, J. Z. Baffi, R. J. C. Albuquerque, M. Nozaki, H. Kaneko, K. Saito, M. G. Rich, M. E. Rothenberg, J. Ambati; Pro-Vascular Mechanisms and in vivo Imaging Strategies of CCR3, a Specific Biomarker for Neovascular AMD. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4303.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Chemokine receptor-3 (CCR3) is a critical regulator of eosinophil migration, yet it also functions as a direct angiogenic modulator on endothelial cells in vitro. We recently discovered that CCR3 is specifically expressed in human choroidal neovascular membranes (CNV) in vivo and that functional blockade suppressed angiogenesis in the laser-injury mouse model of CNV. In this study, we interrogated mechanisms by which CCR3 promotes CNV and developed an in vivo imaging strategy to utilize CCR3 as a novel biomarker in the identification of occult CNV.

Methods: : To determine the pro-angiogenic effects of CCR3 signaling on human choroidal endothelial cells (hCECs), we exposed early passage isolates to specific agonists, eotaxins-1, -2, and -3. Culture responses were evaluated with a fluorescent plate assay for F-actin polymerization and immunoprecipitation for Rac1 activation. For in vivo bio-imaging of CCR3, we identified aged Ccl2/Ccr2 deficient mice, which exhibit neovascular AMD-like features, with occult CNV using ICG angiography. Occult CNV was tested for CCR3 and Ki-67 expression with immunofluorescence. Mice were administered quantum-dot labeled CCR3 or isotype F(ab), created from commercially available monoclonal antibodies, via tail vein injection. Serial fluorescent images were acquired at baseline, 1, 4, 12, and 24 hours after which eyes were harvested and analyzed for CD31, quantum-dot-F(ab) conjugate, CCR3 and Ki-67 expression.

Results: : Stimulation of hCECs with any of the eotaxins induced rapid polymerization of actin molecules, marked cortical F-actin assembly, and Rac1 activation. Occult CNV identified in aged Ccl2/Ccr2 deficient mice expressed both CCR3 and Ki-67. Following injection of labeled CCR3 F(ab), focal branching hyper-fluorescence was observed within 1 hr, while isotype control revealed no detectable signal. On microscopic examination, hyper-fluorescent areas from CCR3 F(ab) injected mice localized to CNV lesions which revealed quantum-dot, CD31, CCR3 and Ki-67 positivity.

Conclusions: : These data demonstrate that CCR3 activation is directly pro-angiogenic and may provide a suitable biomarker for in vivo detection of occult CNV.

Keywords: choroid: neovascularization • imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound) • proliferation 
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