In vivo molecular imaging of VEGFR-2 in VEGF-A–induced angiogenesis. (A) Schematic of our in vivo molecular imaging approach to study VEGFR-2 expression in corneal angiogenic vessels. Fluorescent MSs conjugated with anti-VEGFRs Ab are injected systemically in live animals. (B) α-VEGFRs mAb–conjugated MSs (green) and rhodamin-conjugated conA (red). Arrows indicate MS in blood vessels. Bar, 200 μm. (C) Quantitation of the number of α-VEGFRs Abs-conjugated MS in corneal vessels of untreated and VEGF-A–implanted eyes (day 4; n = 4-12). (D) Distribution of α-VEGFR-2 Ab-conjugated MS or IgG-conjugated MS in VEGF-A–induced angiogenesis (day 4). (E) Representative confocal image (100× objective) of a corneal flatmount on day 6 after VEGF-A implantation. For in vivo molecular imaging of VEGFR-2, animals were injected with α-VEGFR-2 mAb–conjugated MS (green) and 30 minutes later perfused with rhodamin-conjugated conA (red) to stain the vasculature. Bar, 30 μm. (F) Triple staining for LYVE-1 (red), VEGFR-2 (green), and CD31 (blue) in the front of VEGF-A– implanted corneas (day 6). Bar, 100 μm. *P < .05; **P < .01. (G) Angiogenic endothelium expressed higher levels of VEGFR-2 that actively traps VEGF-C on the endothelial surface. Subsequently, the VEGFR-2/VEGF-C complex is internalized and therewith cleared from the extracellular matrix. (H) Mechanism of delayed lymphangiogenesis. Sprouting blood vessel endothelium expresses VEGFR-2 and VEGF-C. Lymphatic endothelium expresses VEGFR-2 and -3 but not VEGF-C. The endothelium of the angiogenic tip VEGF-C is trapped by VEGFR-2 and internalized. In a distance from the angiogenic tips and in absence of the active trapping mechanism, blood endothelial–derived VEGF-C reaches lymphatic endothelium, allowing their growth.