Abstract 2478: Molecular Imaging of Angiogenesis Using Vascular Endothelial Growth Factor-Conjugated Microbubbles
Background: Clinical endpoints in therapeutic angiogenesis trials are problematic due to limited methods to quantify response. Imaging of angiogenesis receptors (REC) could provide a sensitive method for detecting neovascularization. We have shown that microbubbles (MB) tagged with vascular endothelial growth factor-121 (MBVEGF) bind in vitro to the kinase domain VEGF REC (KDR). We tested the hypothesis that MBVEGF bind to angiogenic endothelium in vivo and can be ultrasonically imaged.
Methods: Lipid MB were prepared bearing either human recombinant VEGF (1x105 molecules/MB) or control non-specific IgG (MBCTL). bFGF (3.2ug) was injected into the scrotum of Wistar rats to induce angiogenesis in the cremaster muscle. 7 days later, rats were anesthetized, the cremaster was prepared for intravital microscopy, and 108 MBVEGF (n=8) or MBCTL (n= 8) were injected intravenously (iv). 1 min later, MB adhesion was counted in 20 fields. VEGF REC overexpression was modeled using 6 mice injected subcutaneously with 5x105 squamous cell carcinoma cells. 2 weeks after seeding, 5x106 MBCTL or MBVEGF were given iv. At 4.5 min after injection, low energy harmonic tumor imaging (7 MHz) was performed, immediately followed by a high energy pulse to destroy MB, and resumption of low energy ultrasound at 5 min to detect signal from unbound MB. Tumor videointensity (VI) at 5 min was subtracted from that at 4.5 min to derive VI due to MB adhesion.
Results: On intravital microscopy there was greater microvascular adhesion of MBVEGF (22 ±9) vs MBCTL (4 ±3, p<0.004). Subtracted tumor VI was higher after MBVEGF (17±10) vs MBCTL (8 ±10, p<0.004), with regions of persistent contrast enhancement qualitatively appearing patchy in distribution. Western blot of tumor and bFGF-stimulated cremaster confirmed overexpression of VEGF REC KDR and flt.
Conclusions: MBs bearing VEGF preferentially bind to angiogenic endothelium and can be ultrasonically imaged in vivo. Such use of a naturally occurring ligand as the targeting moiety may confer advantages over conventional antibody targeting approaches, which are immunogenic. Ultrasonic imaging of REC could provide a clinically powerful molecular basis for evaluating therapeutic angiogenesis in patients with ischemic cardiovascular disease.