Ultrasound Molecular Imaging of Angiogenesis Using Vascular Endothelial Growth Factor-Conjugated Microbubbles

Imaging of angiogenesis receptors could provide a sensitive and clinically useful method for detecting neovascularization such as occurs in malignant tumors, and responses to antiangiogenic therapies for such tumors. We tested the hypothesis that microbubbles (MB) tagged with human VEGF₁₂₁ (MB_VEGF) bind to the kinase insert domain receptor (KDR) in vitro and angiogenic endothelium in vivo, and that this specific binding can be imaged on a clinical ultrasound system. In this work, targeted adhesion of MB_VEGF was evaluated in vitro using a parallel plate flow system containing adsorbed recombinant human KDR. There was more adhesion of MB_VEGF to KDR-coated plates when the amount of VEGF₁₂₁ on each MB or KDR density on the plate was increased. MB_VEGF adhesion to KDR-coated plates decreased with increasing wall shear rate. On intravital microscopic imaging of bFGF-stimulated rat cremaster muscle, there was greater microvascular adhesion of MB_VEGF compared to that of isotype IgG-conjugated control MB (MB_CTL). To determine if MB_VEGF could be used to ultrasonically image angiogenesis, ultrasound imaging was performed in mice bearing squamous cell carcinoma after intravenous injection of MB_VEGF. Ultrasound videointensity enhancement in tumor was significantly higher for MB_VEGF (17.3 ± 9.7 dB) compared to MB_CTL (3.8 ± 4.4 dB, n = 6, p < 0.05). This work demonstrates the feasibility of targeted ultrasound imaging of an angiogenic marker using MB_VEGF. This approach offers a noninvasive bedside method for detecting tumor angiogenesis and could be extended to other applications such as molecular monitoring of therapeutic angiogenesis or antiangiogenic therapies in cardiovascular disease or cancer.