Abstract
P 079
Cy5-conjugated Macugen as a novel tool for specifically localizing the VEGF165 protein in choroidal neovascularization membranes
Aysegül Tura1, Sigrid Henke-Fahle2, Olcay Tatar3, Julia Beutel1, Matthias Lüke1, Salvatore Grisanti1
1Universitäts-Augenklinik, Campus Lübeck, Universitätsklinikum Schleswig-Holstein, Lübeck, 2Breuninger Labor, Universitäts-Augenklinik Tübingen, 3Universitäts-Augenklinik Tübingen
Objective
Choroidal neovascularization (CNV) is the principle cause of severe vision loss in age-related macular degeneration (AMD). Despite the experimental and clinical evidence attributing a major role to the VEGF165 isoform in the pathogenesis of CNV, the histological distribution of this protein in CNV membranes (CNVMs) is not determined yet. Here, we utilized Cy5-conjugated Macugen (M-Cy5), a clinically approved RNA-aptamer binding VEGF165, to specifically detect this isoform in CNVMs.
Methods
CNVMs surgically excised from 3 patients with AMD were frozen without fixation. The activated endothelial cells and the total levels of VEGF were detected by immunohistochemistry on serial cryosections using antibodies recognizing CD105 and all VEGF isoforms, respectively. Sections adjacent to the areas of high VEGF immunoreactivity were incubated with M-Cy5 and analyzed by fluorescence microscopy. To enhance the signal intensity, additional sections were incubated with biotin-conjugated anti-Cy5 antibodies and Alexa 488-Streptavidin following M-Cy5.
Results
The membranes exhibited varying degrees of neovascularization, representing diverse stages of the disease. VEGF was present in both the regions of active neovascularisation and in the surrounding stromal cells. The M-Cy5 staining, which appeared more prominent after signal enhancement, localized to areas of strong VEGF-immunoreactivity, particularly around activated endothelial cells.
Conclusions
The high degree of overlap in the signal patterns of M-Cy5 staining and VEGF-immunostaining demonstrates for the first time that the 165-aa isoform constitutes a considerable portion of the VEGF present in the CNVMs. These findings also highlight the potential of Cy5-conjugated Macugen as a useful tool for the histological detection of the VEGF165 protein.
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